National Veterinary Services Laboratories Testing Protocol Real
Contents
1. 7 4 Panigrahy B Senne D A Pedersen J C Avian Influenza virus subtypes inside and outside the live bird markets 1993 2000 a spatial and temporal relationship Avian Dis 46 298 307 8 Summary of Revisions 8 1 The recommended quencher dye was changed from TAMRA to BHQ 1 Cepheid is currently marketing only the Smart Cycler II system which is not calibrated for use with the TAMRA dye as a quencher NVSL AVPRO1510 02 Testing Protocol Page 36 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 8 2 The volume of BHI for the collection of swab pool specimens was changed from 2 0 ml per tube to 2 3 5 ml per tube Section 1 1 8 3 The concentration of Rnase Inhibitor in Table 4a was corrected Version 1510 01 listed the use dilution of Rnase Inhibitor as 6 65 units 50ul Table 4a and the final concentration of Rnase Inhibitor as 0 266 units The correct use dilution of Rnase Inhibitor is 13 3 units ypl The correct final concentration is still 0 266 units pl 8 4 The nucleotide sequence for primer H5 1685 Table 1 was changed from 5 AgA CCA gCT AAC ATg ATT gC 3 to pe AgA CCA gCT ACC ATg ATT gC 3 The change was made to be consistent with that published by Spackman et al J Clin Micro 40 3256 3260 2002 8 5 The Ambion MagMax 96 well magnetic bead RNA isolation procedure was added as an approved method for the extraction of RNA from swab2. Diagnostic specimens which contain inhibitory and should be The bead procedure has been dev that contain inhibitory substances y of the H5 as nt beads are d Ea the matrix pr by rRT PCR All false negative ation The H5 specificity detecting both the NA lineages of H5 AIV ted with a FAM loped to say and to aid in assay istributed by the NVSL CEC imer probe set to screen diagnostic samples because the matrix assay is more broadly reactive positive on detects all subtypes sensitive than the H5 or H7 primer probe assays and is more Samples the matrix assay can be tested to quickly NVSL AVPRO1510 02 Testing Protocol Page 6 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus determine if H5 or H7 subtypes are involved Virus isolation should be performed to confirm the rRT PCR results for index cases involving AIV Tissues from more than one bird should not be pooled together However tracheal swabs from up to 5 birds from a single premise can be pooled together in 2 3 5 ml of brain heart infusion BHI broth but should not be pooled with cloacal swabs Tracheal swabs are the preferred specimen for the isolation of RNA Validation data has demonstrated a decreased efficiency in the isolation of RNA from cloacal swabs due to the heavy load of organic material The LPAI H7N2 virus which is currently circulating in the U S northea
3. The background fluorescence of each lot of probe should be determined following receipt If the background fluorescence exceeds 500 fluorescent units the probe should be discarded and a new lot requested from the manufacturer Probes with high background fluorescence excessive free floating dye will reduce the quantity of fluorescent units available for diagnostic detection The background fluorescence of each assay or run should be monitored to determine if there is an indication of probe degradation Additional information on fluorescent probe handling and storage can found at www operon com www idtdna com and www idahotech com 4 Prepare 70 ethanol with 100 olute ethanol and RNase free water 59 Prepare 80 ethanol with 100 olute ethanol and RNase free water 3 4 6 B Mercaptoethanol 6 ME must be added to the RNeasy RLT lysis buffer before use Add 10ul ME per 1 ml RLT buffer RLT buffer is stable for 1 month after addition of ME Be sure to date buffer after adding B ME 3 4 7 Add 100 absolute ethanol to the RNeasy RPE wash buffer according to the Qiagen RNeasy kit directions 3 4 8 The template for the positive controls is in vitro transcribed RNA from the AIV matrix H5 and H7 protein gene Transcribed RNA is available upon request from the NVSL 1800 Dayton Ave Ames IA Request information is listed below The transcribed RNA positive control should be dil
4. Non optimal storage conditions may lead to degradation of the IC Internal control results should be interpreted as questionable if the IC Cy is higher than 38 The negative no template control is used as the positive control for the IC The Cy value should be evaluated on each run as the stability of the IC could change depending on handling and storage of beads Since the IC and the viral RNA compete for the same set of primers the IC will only be detectable when the viral RNA is either limiting or absent in the sample When the viral RNA has a Ce of 30 or lower viral amplification AIV specific will out compete the IC NVSL AVPRO1510 02 Testing Protocol Page 31 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Table 5a Interpretation of Internal Control Results IC Viral FAM C Interpretation of IC C Interpretation of viral C Texas Red Ci Negativ 27 33 Negative out competed Positive e 38 93 30 91 Negative not valid Positive 35 21 37 59 Positive valid Suspect 35 21 0 00 Positive valid True negative 0 00 0 00 Negative False negative 38 0 or 0 00 No Test non valid C No Test higher The H5 bead reagents will detect both Eurasian H5 subtype viruses for example the Asian H5N1 and North American H5 subtype viruses Virus lineage can not be differentiated with this test 6 Analysis of test re
5. being performed or one plate at a time Beads are only stable when mixed for one month Store at RT Bead Resuspension Mix Volume Per Volume Per Well Plate 1 Bead Resuspension Solution 6 0 pl 750 pl 2 Nuclease free Water 4 0 pl 500 pl Mix briefly then add 3 RNA Binding Beads 4 0 pl 500 pl Mix briefly then add 4 100 Isopropanol 6 0 pl 750 pl Mix well by vortexing Total Volume of Lysis Binding Solution 20 pl 2500 pl 4 3 1 2 Wash Solution I Mix Add 35 ml of 100 isopropanol to Wash Solution I concentrate Mix well and date the bottle to indicate isopropanol was added Store at RT 4 3 1 3 Wash Solution II Mix Add 80 ml 100 ethanol to Wash Solution II concentrate Mix well NVSL Testing Protocol AVPRO1510 02 Page 23 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus and date the label on the box to indicate ethanol was added Store at RT 2 Procedure for Extraction of RNA 4 3 2 1 Vortex swab specimen and transfer 50 pl of sample into the corresponding well on the 96 well processing plate supplied with kit The order of specimens in the extraction plate is stipulated by the order of specimens in the specimen rack Each specimen in the specimen rack is identified with laboratory accession and sample number 4 3 2 2 Place 101 pl of viral lysis binding solution with Poly A RNA to each well contain
6. Figures 2a and 2b The correct value for background maximum cycles is the cycle at the base of the V about 18 for figure 2a Figure 2a Example of a V shaped fluorescence trace The background maximum cycle is set to the default of 40 circled All other analysis criteria are set to the default values The negative control is shown for reference horizontal line at zero Press R key to reset zoom Ctrl A to show all sites Site ID Protocol Samp Click right mouse button to scale graph 4 H9 HK 52 2 5mM H9 HK 52 2 5mM H9 HK 52 3 75 H9 HK 52 3 H9 HK 52 5mM H9 HK 52 5mM A7 H9 HK 52 6 25 Standard SYBRG Melt Fluorescence Views Thresh Manual Thresh Auto Thresh Auto Min Auto Max Valid Min Valid Max Bkgnd Bkgnd Results Table B Setting Fluor Units SD s Cycle Cycle Cycle Cycle Sub Max Cycle Analysis Setti i Manual 30 0 NIA 5 10 3 60 ION 40 Protocols imary Manual 30 0 NIA 5 10 3 60 ION 40 i Manual 30 0 NIA 5 fo 3 60 ON 0 40 Manual 30 0 NIA 5 10 3 60 oN o Figure 2b Same fluorogram as figure 2a however the background maximum cycles have been reduced to 20 circled to align the background fluorescence at 0 units All other analysis criteria are set to the default values The negative control is
7. Personnel handling clinical samples or live virus should take appropriate safety precautions to avoid accidental introduction of the virus Appropriate safety precautions should include wearing disposable gloves laboratory coat and safety glasses as well as handling all specimens in a class II biosafety cabinet NVSL AVPRO1510 02 Testing Protocol Page 7 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 1 2 Keywords Avian Influenza virus AIV matrix gene MA real time reverse transcriptase polymerase chain reaction rRT PCR room temperature RT biological safety cabinet BSC 1 3 Collaboration This protocol was developed in cooperation with the Southeast Poultry Research Laboratory Agricultural Research Service USDA Athens GA 2 Materials 2 1 Facilities equipment instrumentation 2 1 1 Surveillance samples originating from outside a known AIV quarantine zone can be processed in a biosafety level 2 BSL 2 laboratory However samples originating from inside a known AIV quarantine zone or considered suspect for HPAIV should be handled under increased biosecurity This includes restricted access to the laboratory area where the clinical samples are being handled until the samples have been rendered non infectious Once a clinical sample has been treated with lysis buffer for RNA extraction the sample can be moved to a less restrictive environment to complete the RNA extraction and rRT PCR
8. analysis Once a sample is biologically amplified isolated in cell or egg culture and has been confirmed or is suspected of being a HPAI virus the virus should be handled at higher containment level specifically BSL 3 or preferably biosafety level 3 ag h ct w 2 1 2 Class II HEPA filtered biological safety cabinets BSC minimum of 2 preferably 3 with UV germicidal lights are required to maintain sample integrity during processing and testing In addition the Trizol and Qiagen extraction procedures should be performed in a class II BSC that is connected to an external exhaust plenum to minimize exposure to organic chemical fumes 2 1 3 Refrigerator 4 C 2 2 1 4 20 C 3 freezer not frost free NVSL AVPRO1510 02 Testing Protocol Page 8 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 2h SIO Soo freezer 2 1 6 Microcentrifuge non refrigerated International Equipment Co MicroMax Needham Heights MA and refrigerated Hermle Z 360K Germany or Sorval Heraeus Biofuge Fresco Germany 2 1 7 Vortex mixer 2 1 8 Assorted test tubes and Eppendorf tube racks 2 1 9 An integrated DNA RNA amplification and detection instrument system that has the capability to detect specific sequences using hybridization probes Instrumentation should be capable of exciting and detecting fluorescein based probes 450 495nm 5
9. shown for reference horizontal line at zero NVSL AVPRO1510 02 Testing Protocol Page 35 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Press R key to reset zoom Ctrl A to show all sites hi Site ID Protocol Samp Click right mouse button to scale graph i H9 HK 52 2 5mM H9 HK 52 2 5mM __ H9 HK 52 3 75 H9 HK 23 76 H9 HK 525MM H9 HK525mM H9 HK 6216 25 H9 HK 5216 25 Fluorescence Thresh Manual Thresh Auto Thresh Auto Min Auto Max Valid Min Valid Max Bkgnd BoxcarAvg Bkgnfi Bkgnd Setting Fluor Units 5D s Cycle Cycle Cycle Cycle Sub Cycles Min Qfcle Max Cycle f 10 20 40 Manual 30 0 NIA 60 JON 5 al 30 0 _ 6 po 3 feo ON 30 0 NIA 60 ON 0 p 10 0 40 30 0 60 ON 7 References 7 1 Office of International des Epizooties Manual of Standards for Diagnostic Tests and Vaccines Fourth edition 2000 pp 221 222 7 2 Spackman E Senne D A Myers T J et al 2002 Development of a real time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes J Clin Micro 40 3256 3260 7 3 U S Department of Health and Human Services Public Health Service Centers for Disease Control and Prevention and National Institutes of Health Manual of Biosafety in Microbiological and Biomedical Laboratories Fourth edition 1999
10. specimens Equivalency validation conducted at the NVSL demonstrated equivalency between the Ambion MagMax and Qiagen RNeasy procedures 8 6 The manual threshold fluorescence units was lowered from the default of 30 to 25 Section 5 0 The threshold was lowered to increase analytical sensitivity Analytical sensitivity testing to evaluate the change from 30 units to 25 units was conducted at the Southeast Poultry Research Laboratory prior to implementing the change 8 7 Reagent codes and instructions for requesting positive control transcribed RNA were added to section 3 4 8 8 8 Matrix and H5 reagent bead instructions for preparation section 3 5 thermal cycling section 4 5 1 and interpretation of results were included in the prot Reagent beads for the H7 assay are not available at time ocol this
11. 00 550 nm 565 590nm and 630 750nm ranges The current protocol was developed using the Cepheid Smart Cycler Cepheid Smart Cycler P SC2500N1 1 Sunnyvale CA However most if not all of the commercially available real time PCR machines can detect the fluorescein based probes and likely can be used with this test Based on past experience of transferring protocols between different machines changes in cycling times and probe concentrations are often required to get equivalent sensitivity Therefore optimization of the assay on alternative machines is required The optimization data needs to demonstrate that the alternative machine provides comparable sensitivity and limit of detection to the assay described in the current protocol with the Smart Cycler instrument This data should be available for review by APHIS 2 1 10 25y1 Smart Cycler tubes Catalog 900 0022 or 900 0003 Cepheid Smart Cycler Sunnyvale CA 2 1 11 Refrigerated PCR reaction tube holder and mini microcentrifuge to spin reaction tubes Both items are supplied with the Smart Cycler real time PCR system 2 1 12 QiaVac 24 vacuum manifold Qiagen Valencia CA and vacuum pump with a capacity of 18 20 liter min Gast MFG Corp St Louis MO Note Insufficient vacuum pressure may reduce RNA yield and purity The vacuum manifold system with vacuum pump is optional but is a
12. 042 Invitrogen Carlsbad CA Appropriate changes to optimize the protocol for use with alternative reagents or RT PCR kits including cycling paramet ters must be supported by an equivalency evaluation as previously noted 2 2 9 Hydrolysis probes and primers Oligonucleotides Table 1 for the detection of AIV Suggested sources Biosearch Technologies http blackholequenchers com and Operon http oligos qiagen com Suggested primers sources Integrated DNA Technologies http www idtdna com NVSL Testing Protocol AVPRO1510 02 Page 10 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus or Operon can be used to order both primers and probes http oligos qiagen com strongly recommended that the probes and primers be purified to a high reactions HPLC purification as the minimum 2 2 10 Ambion MagMax 96 Viral RNA Isolation Kit 1835 plate kit or 1929 for single tube kit Austin TX 78744 1832 Level to reduce nonspecific Purification is typically performed with Other companies It is Cat Ambion Table 1 AIV real time RT PCR probe and primer sequences Specificity Sequence Matrix any M 25 5 AgA TgA gTC TTC TAA CCg Agg TCg 3 influenza 5 Primer M 64 5 FAM TCA ggC CCC CTC AAA gCC gA TAMRA 3 Probe M 124 5 TgC AAA AAC ATC TTC AAg TCT CTg 3 3 Primer
13. 500 x g for 30 min Collect 250p1 of the tissue supernatant and transfer to a 1 5 microcentrifuge tube 750yl of Trizol LS is added to the tube and sample is vortexed for 15 sec Incubate at room temperature for 7 min 4 2 2 Pulse spin to remove liquid from the tube lid 4 2 3 Add 200p1 100 chloroform to the sample Trizol homogenate Vortex for 15 sec Incubate at room temperature for 7 min 4 2 4 Centrifuge at 12 000 x g for 15 min at room temperature 4 2 5 Transfer 450p1 of the upper aqueous layer to a separate microcentrifuge tube marked with sample number Caution The transfer of organic phase material with the aqueous layer will inhibit the PCR reaction Add 500yu1 of 100 isopropanol Invert tube several times to mix Hold at room temperature for 10 min 4 2 6 Centrifuge at 10 000 x g for 10 min at 4 Cina refrigerated microcentrifuge 4 2 7 Decant liquid Care should be taken to assure that the RNA pellet is not disturbed Add 1 0 ml of 80 ethanol Mix gently 4 2 8 Centrifuge at 10 000 X g for 5 min at 4 C 4 2 9 Decant ethanol Invert tube on a clean tissue wipe and allow to air dry for a maximum of 10 min or until all visible signs of moisture are gone It is NVSL Testing Protocol AVPRO1510 02 Page 21 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 4 3 important not to let the RNA pellet over dry as this will decrease its solubility fr water
14. AVPRO1510 02 Page 1 of 36 National Veterinary Services Laboratories Testing Protocol Real Time RT PCR for the Detection of Avian Influenza Virus and Identification of H5 and H7 subtypes in Clinical Samples Date October 19 2005 Number AVPRO1510 02 Supersedes AVPRO1510 01 September 10 2003 Contact Person Janice C Pedersen 515 663 7551 Approvals s B Panigrahy Date 10 19 05 Brundaban Panigrahy Head Avian Viruses Section s Beverly J Schmitt Date 10 19 05 Beverly J Schmitt Chief Diagnostic Virology Laboratory United States Department of Agriculture Animal and Plant Health Inspection Service P O Box 844 Ames IA 50010 Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA and does not imply its approval to the exclusion of other products that may be suitable NVSL AVPRO1510 02 Testing Protocol Page 2 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Table of Contents 1 Introduction 1 1 Background 1 2 Keywords 1 3 Collaboration Materials 2 1 Facilities equipment instrumentation 2 2 Reagents supplies Preparation for the test Personnel qualifications training Preparation of equipment instrumentation Disinfectants Preparation of reagents control procedures Preparation of samples Preparation of matrix and H5 bead reagents wwwwwuw OU RWNHE Performance of the test 4 1 Extrac
15. H7 1244 5 ATT ggA CAC gAg ACg CAA Tg 3 H7 North 5 Primer American H7 1281 5 FAM TAA TgC TgA gCT gTT ggT ggC TAMRA 3 Probe H7 1342 5 TTC TgA gTC CgC AAg ATC TAT Tg 3 3 Primer H5 1456 5 ACg TAT gAC TAT CCA CAA TAC TCA 3 H5 5 Primer H5 1637 5 FAM TCA ACA gTg gCg AgT TCC CTA gCA Probe TAMRA 3 H5 1685 5 AgA CCA gCT ACC ATg ATT gC 3 3 Primer Refers to the nucl or primer anneals to the AIV genome lidated with the Smart Cycler I system system it is recommended that all hybridizing led with FAM as a reporter dye and quenched with probes were val the Smart Cycler II probes be label either Dabcyl system is not The TAMRA dye or Black Hole quencher I leotide position where the 5 end of the pr obe The FAM TAMRA hybridizing When u The Smart Cycler II sing calibrated to use the TAMRA dye as a quencher dye is read as background noise in channel 2 2 2 11 RNase Inhibitor 40 units pl Promega catalog N2511 or N2515 Madison WI 2 2 12 MgCl 25 mM Promega catalog A3511 or A3513 Madison WLI NVSL AVPRO1510 02 Testing Protocol Page 11 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 2 2 13 TE buffer pH 8 0 1X molecular grade Promega V6231 or V6232 Madison WI 2 2 14 14 3 M 8 mercaptoethanol 8 ME Sigma M 6250 St Louis MO Caution toxic dispense in a fume hood wear
16. Test for Detection of Avian Influenza Virus transferred to NVSL for confirmational testing Suspect samples as well as H5 and H7 positive specimens should be transferred to the NVSL as quickly as possible for confirmation and further testing Additional samples may need to be collected for further testing All suspect specimens and those only positive by the matrix rRT PCR should be confirmed by virus isolation before reporting as positive Any questionable samples should be re tested If results of the second test are unsatisfactory additional sampling from the flock or premises should be considered if possible Confirmation by virus isolation is required before a foreign animal disease FAD will be officially diagnosed 6 2 Expected C for the transcribed RNA positive control The transcribed RNA positive control should be diluted to a working concentration that will consistently give a target C of approximately 25 0 Any test run where the positive control has a Cy higher than 29 0 should be repeated to assure that test reproducibility is maintained If the C of the positive control consistently runs lower than 20 0 recalibration dilution of the positive control is suggested Any time the positive control or any other growth curve Cy is lower than 14 0 the background subtraction can be skewed 6 3 Recommendations for evaluating fluorograms Evaluation of the fluorogram with the foll
17. ackhole quencher BHQ 1 at the 3 end The proximally located quencher dye absorbs the emission of the reporter dye as long as the probe is intact and not hybridized to the target When the probe is hybridized to the target the 5 nuclease activity of Taq polymerase will cause hydrolysis of the probe separating the quencher from the reporter dye This separation results in an increase in fluorescence emission of the reporter dye which is detected spectrophomectrically and recorded The Testing Protocol AVPRO1510 02 Page 5 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus amount of fluorescence recorded is proportional to the amoun t of target template in the samples The AIV rRT PCR procedure described in this protocol has three unique primer probe combina tions one that targets the matrix gene and two that are subtype specific for H5 and H7 AIV s a primer probe sets are designed H5 and H7 viruses In to successfully detect gene assay has a detec 1 000 copies of target infectious dose of sets each have a de approximately 10 to 10 sensitivity and specifi to virus isolation was respectively based on virus tection 4 whereas the The matrix primer probe set is designed to detect ll influenza A viruses H5 and H7 subtyping det the Lo addit
18. and Briefly vortex 4 2 10 Hydrate pellet in 50ul of RNas incubate at 4 C for 1 hr or overnight to resuspend pellet before pipetting High throughput magnet bead RNA extraction from swab specimens Ambion MagMax method This procedure describes the extraction of total RNA from swab specimens using the Ambion extraction reagent MagMAX Viral throughput purification of total oropharyngeal tracheal cultured cells rapidly disrupts cellular membranes nucleases surface are added beads The RNA Isolation Kit is designed for rapid high RNA from and cloacal swab samples as well The addition of guanidinium thiocyanate and inactivates cellular a nucleic acid binding bind nucleic acids The retained with the use of as Paramagnetic beads with to the lysate to the RNA are then containing magnets and the supernatant containing cell debris and other contaminants is removed during the wash procedures Different systems for RNA isolation are commercially available and potentially may work as well procedure as the described can be substituted when Alternative methods comparison testing of new and standard methods show equivalency This data should be available for review by APHIS 4 3 1 Preparation of Ambion Viral Isolation Kit Components Lysis Binding Solution See table below to determine the quantity of lysis binding solution nee
19. anded RNA It contains sequences complementary to AI matrix forward primer at the 5 end and AI matrix reverse primer at the 3 end and has a unique internal seguence for binding to the IC probe The matrix bead includes a FAM labeled probe for detection of the viral matrix gene and the Cal Flour Red 610 labeled probe for the IC NVSL AVPRO1510 02 Testing Protocol Page 12 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus ms The H5 reagent bead includes two forward primers one for the detection of North American NA lineage H5 AIV and the other for the detection of the Eurasian EA lineage H5 AIV The H5 bead includes one FAM labeled H5 probe that will detect both the NA and EA amplicon The beads are distributed in 0 5 ml micro centrifuge tubes which can be used for reconstitution of the reagents The beads are to be used with Qiagen One Step RT PCR dNTP and enzyme The beads can be stored in the foil packaging at 4C for 6 months Do not E y remove from foil packaging till ready for use s Refer to the MSDS before Use caution when using these reagent individual Material Safety Data Sheet handling any of these reagents E E p 3 Preparation for the test For this procedure it is critical to have separate preparation areas and equipment for nucleic acid extraction clean procedures an
20. d at the cycle where the primary curve crosses the threshold Usage Accept the default setting Assay for the FAM channel Background Subtraction Accept the default value of ON xg Background Minimum accept the default value of 5 Min The first cycle used to calculate the background if the background subtraction is ON Testing Protocol AVPRO1510 02 Page 29 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Background maximum cycle Enter for the calculation of the background if subtraction is ON The default Threshold Settings accept the default of is 40 Manual Threshold Fluorescence units Enter 2 28 as the maximum cycle the background Manual 5 fluorescent units It is critical that the C be above the background fluorescence The closer the threshold is set to the background fluorescence the more sensitive the detection limit However if the threshold is set too close to the background fluorescence background noise could cross the threshold and be reported incorrectly as a positive sample By lowering the threshold fluorescence units from 30 default value to 25 the analytic sensitivity of the assay is increased The possibility of reporting a false negative is reduced while the possibility of detecting a false positive is increased Boxcar Av
21. d more than 4 times A total of 3 pmol of the matrix H5 and H7 probe are added respectively per 25 pl reaction 3 4 3 Handling and Dilution of Primers and Probe Lyophilized primers and probes must be centrifuged briefly to ensure that the DNA pellet is at the bottom of the tube before they are opened and reconstituted TE buffer should be used for the initial reconstitution of lyophilized primers and probes Quantitation information will be supplied for each oligonucleotide primer oligo by the manufacturer An example of calculation for oligo reconstitution You have 17786 pmol of oligo will be on oligo information sheet from manufacturer Need 200pmol yl for stock concentration Divide pmol of oligo by the pmol pl needed or 17786 pmol 88 9ul of 1X TE 200 pmol ypl The calculation for the probe is the same except divide the number of probe pmol by 120 pmol yl Mix gently by tapping the tube and allow the oligo to rehydrate for about 10 minutes before us Working stocks of primers should be 20 pmol pl 20uM and working stocks of probes should be 6 pmol pl NVSL AVPRO1510 02 Testing Protocol Page 16 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Dilute the primers 1 10 and dilute the probe 1 20 in nuclease free H20 do not use TE buffer for the working stocks
22. d work with amplified nucleic acid The clean area is used for preparing reagents for the PCR procedure Amplified c DNA or sample RNA should never be introduced into this area One biological safety cabinet should be designated for clean work only There should also be a separate set of clean pipettors and tips RNase free water tubes for reagent preparation racks and ice container which are designated for clean use only and never leave the area A 20 C freezer should also be designated as clean for storage of reagents A second biological safety cabinet set of pipettors and other equipment and reagents should be used for extraction procedures Ideally a third biological safety cabinet should be used for transfer of RNA to amplification tubes Latex nitrile gloves in particular must be worn throughout the procedure and must be changed frequently RNA is very labile and easily degraded by RNases that are ubiquitous including on human skin Gloves also help protect the reagents and samples from other contaminating agents and cross contamination that can adversely affect results Always change gloves after working with sample RNA or amplified DNA Always wear fresh gloves when working with clean reagents Protective eyewear gloves and lab coats should be worn as some of the reagents used are toxic It is recommended that the Qiagen and Trizol extraction procedures be conducted NVSL AVPRO1510 02 Testin
23. ded Add 1 0 pl Poly A RNA kit provided per 50 pl lysis binding and mix briefly Following the addition of Poly A RNA add 50 pl of 100 isopropanol to lysis binding Poly A mixture to give a total volume of 101 pl per sample Vortex well lLysis binding solution is stable at room temperature for one month It is not recommended to store the prepared Viral Lysis Binding Solution at 4 C or below as this may cause the carrier RNA to precipitate if the solution is inadvertently stored at 4 C warm it at 37 and shake to dissolve any precipitates before us NVSL AVPRO1510 02 Testing Protocol Page 22 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Add carrier RNA to Lysis Solution and mix well before adding isopropanol or carrier RNA may be very difficult to disperse l Comoine the collllowaing 1 well 1 Plate 4 Plates Viral Lysis Binding Soln 50 0 pl 6 25 ml 25 0 ml Concentrate Carrier RNA 1 0 ul 125 pl 500 wl 2 MIX WELL then add 100 Isopropanol FUS pa 6 25 ml 25 0 ml 3 MIX WELL by vortexing 4 3 1 1 Bead Resuspension Mix Dilute the Bead Resuspension Solution with Nuclease free Wateraccording to the table below Be sure to add 100 isopropanol to the mixture last or beads may clump together and be more difficult to enter into solution Prepare only enough for the number of extractions BT a
24. e silica gel membrane with the pipette tip Incubate at room temperature for at least 1 minute Elute RNA by centrifuging for 1 minute at gt 10 000 rpm Discard RNeasy column 4 1 10 Store at 4 C until specimen is tested on rRT PCR RNA should be stored at 4 C for as short of period as possible before testing If the sample cannot be tested within 24 hours it should be stored at 20 C or colder Trizol LS Extraction for tissue samples This procedure describes the extraction of total RNA from tissue using the Trizol extraction reagent The reagent is a mono phasic solution of phenol and guanidine isol cen thiocyanate Addition of chloroform followed by trifugation separates the solution into an aqueous phase NVSL AVPRO1510 02 Testing Protocol Page 20 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus and an organic phase The RNA is recovered from the aqueous phase by precipitation with isopropanol Different systems for RNA isolation are commercially available and potentially may work as well or better than the method described here Alternative methods of RNA extraction can be substituted if testing shows equivalency This data should be available for review by APHIS Always wear protective clothing eyewear and latex gloves when working with extraction reagents 4 2 1 Centrifuge tissue specimens 10 20 homogenates or tissue pools at 1
25. erage accept the default of 0 5 1 Verification of test results The only changes from the Cepheid Smart Cycler default were the Max background and threshold settings Wi analysis settings a specimen will be called positive crosses the C when the fluorescen th these ce units exceeds 25 units These settings were designed to optimize the discrimination of positive and negative specimens for this sults from each run still particular protocol but the re need to be verified by the user log linear phase when crossing The curve sho the Max background the incidence the is reduced For samples that s Eid threshold uld be in the By lowering have a v of traces with a V shape shaped trace the Max background setting can be incrementally lowered to 15 until the early cycles in the trace are approximately horizontal and aligned with zero f LuOrescence Figures 2a and 2b When lowering the Max background keep in mind that you want as many cycles as possible used to calculate the background subtraction This is a correction o software handles the data and it decreases th cycles used to calculate the background and correct the curve If you have questions about the trace trace with the background subtraction off n how the number of look at the This shows the raw fluorescence data a
26. g Protocol Page 13 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus in a vented class II BSC NVSL Testing Protocol Real Time RT PCR Test AVPRO1510 02 Page 14 of 36 for Detection of Avian Influenza Virus 3 1 Personnel qualifications training Personnel performing PCR procedures should be familiar with 3 1 1 Preparation and proper handling of samples and reagents 3 1 2 Calibration maintenance and use of instruments included in this protocol 3 2 Preparation of equipment instrumentation Refrigeration equipment incubators centrifuges pipettors and thermal cyclers are calibrated and certified according to the respective institution standard operating procedures 3 3 Disinfectants Several classes of disinfectants e g iodophores phenolics quaternary ammonia compounds 70 alcohol 10 sodium hypochlorite inactivate AIV by destroying the lipid envelope of the the disinfectants listed above only virus However of sodium hypochlorite Vircon S have been oe and peroxigen compounds will bleach and peroxigen compounds shown to degrade nucleic acid as well as destroy infectivity of AIV This is important when selecting a disinfectant to rid surfaces of contaminating nucleic acids 3 4 Preparation of reagents control procedures 3 4 1 Oligonucleotide primers Prepare primers in a clean hood see 3 0 Always wear latex nitrile
27. gloves 2 2 15 Sterile aerosol resistant pipette tips of various sizes 1 0ml 200 50p1 100 10p1 10 0 5p1 2 2 16 1 5 ml microcentrifuge tubes sterile 2 2 17 Powder fr latex or nitrile gloves 2 2 18 Calibrated pipettors from 0 5 pl to 1000 ul 2 complete sets and one extra pipettor 5 40 ul for DNA transfer 2 2 19 Ambion Magnetic Stand 96 Ambion Inc catalog 10027 Austin TX or an O ring 96 well Magnetic Ring Stand Cat 10050 Ambion Austin TX Product 10050 has 96 ring magnets that pellet beads in a donut shape Product 10027 has 24 big magnetic rods that pellet beads to one side of the wells 2 2 20 Orbital shaker for 96 well plates Lab Line Titer Plate Shaker Model 4625 Melrose Park ILL 2 2 21 5 250 pl 12 channel pipetting tool Matrix Technologies Corp catalog 2012 Hudson NH 2 2 22 Lyophilized AIV rRT PCR matrix and H5 reagent beads have been prepared by Cepheid Sunnyvale CA for use with the matrix and H5 assays Request for reagent beads should be submitted to the NVSL Each bead is sufficient for four 25 pl reactions Each bead consitists of assay specific primers and probe KCl MgCl2 and HEPES buffer In addition the matrix bead includes an internal control IC The purpose of the IC is the detection of a false negative resulting from non specific inhibition of PCR amplification The matrix IC is 228 bases long in vitro transcribed single str
28. gloves when dispensing primers Dilute primers to 200 pmol pl 200uM in 1X TE for the tock dilution and 20 pmol l in RNase free water for he working dilution Aliquot primers in small volumes UPU tU o avoid excessive freeze thaw cycles For short term torage of primers lt 2 week 4 C is acceptable For onger term storage 20 C or colder is recommended tore stock primer solutions at 20 C or 70 C 10 pmol of the matrix forward and reverse primers and 10 pmol of both the H5 and H7 forward and reverse primers in the subtyping assay are added per 25 pl reaction See section 2 2 9 NVSL AVPRO1510 02 Testing Protocol Page 15 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 3 4 2 Hydrolysis Probe Prepare probe in a clean hood see 3 0 Always wear latex nitrile gloves when working with hydrolysis probes Hydrolysis probes are light sensitive and should be protected from exposure to direct light Diluted probes should be stored in amber sterile RNase free microcentrifuge tubes or tubes wrapped with foil Dilute probes to 120 pmol pl 120uM in 1X TE for the stock dilution and to 6 pmol pl in RNase free water for the working dilution Aliquot probe in small volumes to avoid excessive freeze thaw cycles Store diluted probes at 20 C and stock probe solutions at 20 C or 70 C Avoid excessive freezing and thaw cycles Diluted probe should not be frozen thawe
29. highly recommended product for processing large numbers of samples for rRT PCR NVSL AVPRO1510 02 Testing Protocol Page 9 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 2 2 Reagents supplies 2 2 1 Molecular biology grade RNase free sterile distilled water 2 2 2 In vitro transcribed AIV matrix gene positive control RNA and H5 and H7 In vitro transcribed control RNA supplied by the NVSL Ames IA 2 2 3 Ethanol absolute Caution irritant flammable 2 2 4 Tsopropanol 99 pure Caution irritant flammable 2 2 5 Chloroform 99 pure Caution toxic 2 2 6 Trizol LS reagent Caution toxic in contact with skin and if swallowed causes burns Cat 10296 010 or 10296 028 Invitrogen Carlsbad CA 2 2 7 Qiagen RNeasy Extraction Kit Cat 74104 50 preps or 74106 250 preps Qiagen Valencia CA 2 2 8 One Step RT PCR Kit Cat 210210 or 210212 Qiagen Valencia CA Many one step RT PCR kits are commercially available However the Qiagen system has been tested extensively with good results Other kits can be considered for use but a minimum level of equivalency testing is required before substituting any reagents in the approved protocol Currently the only alternative kit that has been tested that has comparable test results to the Qiagen system is the Superscript One Step RT PCR System with Platinum Tag DNA Polymerase Cat 10928 034 or 10928
30. ing sample After addition of the lysis binding solution the processing plate may be removed from the BSC All remaining steps can be performed on the deck at room temperature Shake plate on the plate shaker at approximately 550 600 rpm for 30 sec 4 3 2 3 Add 20 pl of RNA Binding Beads mix to each well Shake plate on orbital shaker for 4 minutes at approximately 550 600 rpm 4 3 2 4 Capture pellet the RNA Binding Beads on a magnetic stand Pellet the beads on the Ambion Magnetic Stand 96 for 2 minutes The mixture should become transparent indicating the capture is complete Remove supernatant from beads with plate still on magnet Discard the supernatant 4 3 2 5 Remove the plate from the magnetic stand 4 3 2 6 Add 100 pl Wash Solution I Mix with isopropanol added to each well Shake for 30 seconds at approximately 550 660 rpm The RNA Binding Beads may not fully disperse during the step this is expected and it will not affect RNA purity or yield 4 3 2 7 Pellet the beads for 1 minute and remove supernatant as in step 4 2 2 4 NVSL Testing Protocol 4 4 AVPRO1510 02 Page 24 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 4 3 2 8 Add 100 pl Wash Solution II Mix with ethanol added to each well at approximately 550 600 rpm Shake for 30 seconds 4 3 2 9 Pellet the beads for 30 seconds and remove super
31. ion the Asian H5N1 ion limit of RNA and can de The H5 an limit of gene copies city of the ma shown to be 8 the testing of N LU 1A LUY ect most North American H5 assay has been shown virus The matrix fg or approximately tect 0 1 50 egg d H7 specific probe 0 fg of target RNA or The diagnostic trix assay as compared 8 2 and 99 5 gt 3 500 samples collected during the 2002 outbreak of H7N2 AIV in Virginia When th samples from th same samples were compared by submission the s In addi same premises specificity was shown to be 95 1 and 99 1 tion to the standard wet mix rRT PCR procedure all ensitivity and respectively an alternative reagent bead procedure for use with the matrix and H5 assays has been includes an internal co forward and reverse primers as the viral Th whi described ntrol IC ific detected with an IC sequenc probe substances give false negative results 1 77 no test reported as a specimens should be tested by virus isol reagent bead has dual Eurasian EA labeled H5 HA probe to identify specimens increase the specificit standardization Reage as a non catalog reagen It is preferable to use and North American Both the EA and NA primer sets are de Sp e matrix reagent bead ch utilizes the same target but is Texas Red 5 labeled
32. natant Discard the supernatant Remove the processing plate from the magnetic stand 4 3 2 10 Repeat steps 4 2 2 8 4 2 2 9 to wash a second time with Wash Solution II 4 3 2 11 Shake vigorously for 2 minutes shaker dial position 9 Lab Line to briefly dry the beads It is important to remove residual e samples Residual ethanol may affect RT PCR efficiency If necessary shake longer than 2 minutes to dry but do not shake minutes total thanol from the longer than 5 0 4 3 2 12 Add 50 pl of Elution Solution RT and shake for four minutes at approximately 1000 rpm 4 3 2 13 Pellet the beads for 2 minutes and transfer the RNA into a sample tube kit provided or rRT PCR amplification plate storage plate 4 3 2 14 Store at 4 C until specimen is tested on 4 C for as short rRT PCR RNA should be stored at test of period as possible befor ting If the sample cannot be tested within 24 hours it should be stored at 70 C Reverse transcription and PCR Two work areas are required for this procedure a clean area with a dedicated BSC the freezer and supplies anda thermal cycling area Never introduce RNA DNA material into yN J clean area and always change gloves befor ntering the yN 7 clean A area 4 4 1 In the clean hood prepare a master mix of the following reagents sufficient samples being
33. nd can sometimes aid interpretation NVSL AVPRO1510 02 Testing Protocol Page 30 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Background fluorescence should be monitored routinely Probe degration is indicated by an increase in background fluorescence The fluorescence trace of each specimen should be reviewed before accepting the Smart Cycler positive negative result Any specimen whose fluorescence trace has a gradual increase in fluorescence units and approaches but does not cross the Ce Should be sent to the NVSL for further testing These specimens are referred to as late risers LR and on some occasions may indicate a sample near the threshold of detection At this time the significance of late riser samples is not known and testing by virus isolation is recommended 5 2 Interpretation of internal control results matrix bead Assay systems for the bead reagents were optimized with the Cepheid Smart Cycler II system The results of amplification of viral RNA should be viewed on FAM channel and that of IC on Texas Red ROX channel emission 606 650 nm The Cy values corresponding to the IC should read between 34 and 38 valid range in the absence negative control or presence of template RNA The IC Cy value for the valid range may increase depending on storage conditions and time The beads should be stored in the foil packet at 4C for a maximum of 6 months
34. on portion of the tube may be an indication of insufficient volume of RT PCR master mix or the absence of sample RNA 4 4 6 Insert reaction tubes into thermal cycler and select the designated PCR run protocol start run and enter sample identification as well as positive and negative control information into the sample identification portion of the results table Save run NVSL Testing Protocol AVPRO1510 02 Page 27 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Table 4a conditions for primer probe set MA gene Real time RT PCR reaction mix volumes and type A influenza H5 and the H7 Volume Per Final Reaction Concentration H20 6 95 ul 5X buffer 5 1X 25mM MgCl gt 2 L25 3 75 mM dANTP s 10 mM 0 8 320 uM ea dNTP each Forward Primer 0 5 10 pmol 25y 20 pmol ul Reverse Primer 0 5 10 pmol 25y 20 pmol ul Rnase Inhibitor 0 5 0 266 units pl 13 3 units ul Enzyme Mix x0 Probe 0 5 0 12 uM 6pmol ul MM per rxn 17 Template 8 Total 25ul xQiagen buffer already contains 2 5 mM MgCl at 1X concentration 4 5 Reverse Transcription and PCR for Matrix and H5 bead assay 4 5 1 Preparation of matrix and H5 bead reagents Each bead is sufficient for four 25 ul reactions Reconstitute each bead following RNase fr dNTP Mix Enzyme Mix RNase Inhibitor Total water From Qiagen OneStep RT PCR F
35. owing conditions may be helpful in determining results manually Importantly each specimen should be analyzed independently 6 3 1 Identification of weak positives Remove all reactions with greater than 100 units increase in fluorescence from the graph this changes the scale making it easier to identify weak positives Figures la and 1b Looking at each sample alone will also help to identify weak positive reactions NVSL AVPRO1510 02 Testing Protocol Page 33 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Figure la Example of a fluorogram from samples run on the Smart Cycler Background subtraction is on All analysis criteria are set to the customized default values Note that scale is from 0 to 1000 fluorescence units Y axis making it difficult to evaluate weak positive samples Figure 1b Same fluorogram as figure la however all samples which increased greater than 100 units in fluorescence were removed from the graph Note that the scale is from 0 to 120 fluorescence units Y axis making it easier to recognize weak positives NVSL AVPRO1510 02 Testing Protocol Page 34 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 6 3 2 If there are samples which have a V shaped fluorescence trace lower the background maximum cycles analysis settings table until the early cycles are approximately horizontal and aligned with zero fluorescence
36. plied 5X reaction buffer kit supplied dNTP s 25mM MgCl2 and forward and reverse primers into a nuclease free microcentrifuge tube using the volumes per reaction for each reagent given in table 4 Frozen reagents including MgCl2 should be briefly vortexed and pulsed centrifuged prior to pipetting Next add the RNase inhibitor and enzyme Add the probe last Mix reagents and centrifuge briefly Once the probe has been added to the reaction mix minimize exposure to light ct h ct NVSL AVPRO1510 02 Testing Protocol Page 26 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 4 4 3 Move the master mix to the RNA transfer hood and add the reaction mix 17ul to the Smart Cycler tubes add the mix to the bottom of the cup at the top of the reaction tube 4 4 4 Add 8 0 pl of template RNA to the Smart Cycler reaction tube using a pipettor designated for RNA transfer Close the lid of the tube and number the reaction tubes according to test worksheet Transfer 8ul of diluted transcribed RNA into the positive control reaction tube and 8ul of clean RNase free water into negative control reaction tube The transcribed RNA should be diluted by the user to a working dilution that will have a cycle threshold of approximately 25 0 4 4 5 Centrifuge reaction tubes briefly to remove any air bubbles from the reading window of the PCR tubes Bubbles in the reacti
37. propriate tissue spleen lung intestine should be processed by preparing a 10 20 tissue homogenate and extracting RNA using the Trizol extraction procedure Alternatively 5mm piece tissues are added to 2 0 ml of brain heart infusion broth BHI frozen solid thawed and centrifuged The supernatant from this tissue pool 250 n1 is extracted using the Trizol procedure Validation equivalency data indicate that cloacal swab specimens are less sensitive than oropharyngeal tracheal swabs for the detection of AIV by rRT PCR The Ambion and Trizol extraction procedures are more sensitive for the isolation of RNA from cloacal swab pools 5 swabs pool than the Qiagen extraction procedure Cloacal swab specimens testing negative by rRT PCR must be tested by virus isolation to determine if the specimen is negative for AI However if there is sufficient virus in the cloacal swab specimen it may be detected by rRT PCR Environmental swabs are inappropriate specimens for rRT PCR and should be tested NVSL AVPRO1510 02 Testing Protocol Page 18 of 36 4 tips Real Time RT PCR Test for Detection of Avian Influenza Virus by virus isolation Tissues from more than one bird should not be pooled together All samples should be processed in a Class II BSC Samples received for testing are compared to accompanying paperwork
38. rom Promega Cat N2511 AI matrix or H5 with the 60 0 pl Seed Ud 4 0 pl 2 0 pl 26 units 68 0 pl Kit Cat 210210 or 210212 NVSL AVPRO1510 02 Testing Protocol Page 28 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Distribute 17 pl of the above mix into 4 Smart Cycler reaction tubes and add 8 ul of template viral RNA Centrifuge the tubes in Smart Cycler centrifuge 4 5 2 Thermocycling Conditions for bead reagents RT Step 1 cycle 30 min 50 RT Step 15 min 95 Cc Probe Primer Step Time Temp set AIV matrix 40cycles denaturation 20 sec 94 C annealing 20 sec 60 Cc H5 40 cycles denaturation 1 sec 94 C annealing 20 sec 54 C extension 15 sec 724C 5 Data Analysis Settings for the Cepheid Smart Cycler The Smart Cycler software provides multiple methods for determining the cycle threshold C All data analysis options discussed below are selected from the Analysis Settings from the View Results screen Changes in the standard default settings have been made to customize the analysis for the detection of AIV with the matrix H5 and H7 primer probe sets Edit the Smart Cycler default analysis settings as described below so the raw data will be analyzed according to the analysis parameters described Curve Analysis Accept the Primary curve setting The Cy is detected and reporte
39. stern live bird market system is inconsistently shed in the feces Therefore it is often not possible to detect the virus in the feces or cloacal swabs by virus isolation or rRT PCR It should be emphasized that the rRT PCR technique will detect viral nucleic acid from infectious as well as noninfectious virus The rRT PCR is not the test of choice to determine if infectious AIV is present in environmental samples The procedure described here is used in the Diagnostic Virology Laboratory DVL of the National Veterinary Services Laboratories NVSL The brands of equipment listed in the protocol are used in the DVL however comparable equipment may also be used Laboratories using this protocol should follow quality assurance procedures as they pertain to equipment maintenance receiving specimens and recording reporting results Any specimen from outside a USDA quarantine zone that is positive with the AIV matrix assay regardless of the H5 or H7 assay results should be referred to the National Veterinary Services Laboratories for further testing and characterization Virus isolation and characterization is required to officially diagnose a foreign animal disease FAD such as HPAI There is recent evidence that some strains of ATV can cause infections in humans resulting in conjunctivitis and in rare cases illness and death
40. sults 6 1 Specificity of matrix H5 and H7 assays The matrix AIV assay is designed to detect all subtypes of AIV whereas the H5 and H7 subtype specific assays were designed to aid in the rapid identification of North American strains H5 and H7 AIV The H5 assay has been demonstrated to successfully detect the Asian strain of H5N1 AIV Because the matrix AIV test is more sensitive approximately 10 fold than either the H5 or the H7 the matrix AIV test is used to screen all samples Samples positive by the matrix and either of the H5 or H7 assays provides strong evidence that the sample contains AIV RNA However samples positive by the matrix rRT PCR but negative by the subype specific H5 and H7 assays could still contain H5 or H7 RNA if the quantity of RNA is below the detection limit of the assays or if a subype other than H5 or H7 is causing the infection These specimens should be considered to be suspect samples The matrix H5 and H7 assays have been designed to err on the side of detecting false positive specimens Asa result specimens with a Cr of 35 or higher are considered suspect positives Samples with a Cy of 35 or higher should be repeated on the rRT PCR If positive results including those with C s above 35 are obtained with repeat testing the original specimen should be NVSL AVPRO1510 02 Testing Protocol Page 32 of 36 Real Time RT PCR
41. t has been marked to identify the specimen Centrifuge for 15 sec at gt 8 000 X g at RT Check to assure th ntire specimen has NVSL AVPRO1510 02 Testing Protocol Page 19 of 36 4 2 Real Time RT PCR Test for Detection of Avian Influenza Virus flowed through the column Repeat until all of specimen has been applied to the column Alternatively a QiaVac manifold can be used to pull the specimen and wash solutions through the collection columns This will increase the efficiency and eliminate the need to centrifuge the columns at the steps 4 1 3 4 1 4 4 1 5 4 1 6 and 4 1 7 4 1 5 Add 700pn1 of RW1 buffer to the RNeasy column and centrifuge for 15 sec at gt 8 000 x g and place the column in a clean collection tube the tube with RW1 flow through may be discarded 4 1 6 Add 500u u1 RPE buffer to the RNeasy column and centrifuge for 15 sec at gt 8 000 x g Discard flow through from the collection tube 4 1 7 Repeat for a total of 2 washes with RPE buffer discarding flow through from the collection tube Following the last RPE wash place the RNeasy column in a new 2 ml collection tube 4 1 8 Centrifuge the empty RNeasy column an extra 2 minutes at full speed and discard the collection tube 4 1 9 Place the RNeasy column in an elution tube or a 1 5 ml microfuge tube that has been marked with the specimen number and add 50ul RNase free HO to the column Do not touch th
42. tested The amount per sample This procedure was designed for Cycler Cepheid Sunnyvale CA for the number of t given in the table is pi ay the Cephied Smart NVSL AVPRO1510 02 Testing Protocol Page 25 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus Information on setting up and programming the Smart Cycler can be found in the Smart Cycler user s manual The conditions for the RT PCR on the Smart Cycler are shown in tables 2 and 3 Table 2 RT step thermocycling for Qiagen one step RT PCR Kit RT Step t eyele 30 Mats 50 C 15 min 95 Table 3 Thermocycling conditions for gene specific probe and primer sets Probe Primer Step Time Temp set AIV matrix 45 cycles denaturation A SEC 94 C annealing 20 sec 60 C H7 40 cylces denaturation 1 sec 94 C annealing 20 sec be CG H5 40 cycles denaturation 1 sec 94 C annealing 20 sec 57 C extension 5 sec 72 C Note The fluorescence is detected at the annealing step 4 4 2 The real time RT PCR reaction should be prepared with the following components and volumes using the appropriate primer and probe set and cycling conditions Set up the reactions with the reaction tubes in the cooling block and use aerosol resistant pipet tips Prepare the reaction mix everything but the template by pipetting H20 kit sup
43. therefore the detection and or isolation of H5 or H7 subtype is considered serious Depending on the age and type of bird and on environmental factors clinical signs for HPAI may vary from sudden death to characteristic respiratory signs including excessive lacrimation Sinusitis diarrhea and edema of the head wattles and comb However the vast majority of AIV isolates are low pathogenic AIV LPAI which may range from no obvious clinical signs to a mild respiratory disease Therefore the clinical signs for HPAI and LPAI are not considered pathognomonic Due to the risk of a low virulent H5 or H7 becoming virulent by mutation in poultry hosts all H5 and H7 viruses have also been classified as notifiable avian influenza NAI Highly pathogenic AIV as well as all H5 and H7 viruses are reportable to the World Organization for Animal Health OIE The real time reverse transcriptase polymerase chain reaction rRT PCR technique was developed to assist in the rapid diagnosis of avian influenza infections in poultry The technique utilizes a one step protocol with specific primers designed to amplify a portion of the genome that contains a target PCR sequence Non extendible fluorogenic hydrolysis Tagqman probes monitor the target PCR product formation at each cycle during the PCR reaction The probes are labeled at the 5 end with a reporter dye e g FAM and a quencher dye e g bl
44. tion of RNA from swab specimens Qiagen RNeasy method 4 2 Trizol LS extraction for tissue samples 4 3 High throughput magnetic bead RNA extraction from swab specimens Ambion MagMax method 4 4 Reverse transcription and PCR 4 5 Reverse transcription and PCR for matrix and H5 bead assay Data Analysis Settings for the Cepheid Smart Cycler 5 1 Verification of test results 5 2 Interpretation of internal control results matrix bead Analysis of test results 6 1 Specificity of matrix H5 and H7 assays 6 2 Expected C for the transcribed RNA positive control 6 3 Recommendations for evaluating fluorograms NVSL AVPRO1510 02 Testing Protocol Page 3 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 7 References 8 Summary of Revisions NVSL AVPRO1510 02 Testing Protocol Page 4 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus 1 Introduction 1 1 Background Avian Influenza AI is an economically important disease of chickens and turkeys and many other species The etiologic agent of AI is the avian influenza virus AIV a member of the family Orthomyxoviridae genus Influenzavirus A There are currently 16 antigenically different hemagglutinin H and nine antigenically different neuraminidase N subtypes recognized each virus possessing one H and one N in any combination Of the 16 H subtypes of AIV only H5 and H7 viruses of certain lineages have caused HPAI in poultry
45. to assure correct samples were received and that appropriate samples were submitted for the PCR test Cases are logged into the logbook 3 6 Preparation of matrix and H5 bead reagents Each bead is sufficient for four 25 pl reactions Reconstitute each bead AI matrix or H5 according to the instructions in section 4 5 The reagent beads do not include dNTP s RNase inhibitor or RT PCR enzymes Qiagen enzyme mix and dNTP s as well as RNase inhibitor will need to be provided by the user Performance of the test Before beginning the RT PCR test place clean pipettors racks etc into the clean hood Similarly place the sample equipment into a separate hood Exposure to UV germicidal light for several hours or overnight may be beneficial for the degradation of contaminating RNA from pipettors and other equipment 4 1 Extraction of RNA from swab specimens Qiagen RNeasy method 4 1 1 Vortex swab specimen fluid and transfer 500pl1 of sample into the microcentrifuge tube labeled with the specimen number 4 1 2 Place 500 pl of Qiagen buffer RLT with ME into the microcentrifuge tube Vortex well 4 1 3 Pulse spin to eliminate liquid specimen in the lid after vortexing Add 500p1 70 ETOH and vortex well Centrifuge lysed swab specimen for 5 min at 5 000 X g in a microcentrifuge at RT 4 1 4 Transfer all of the lysed specimen supernatant to a RNeasy Qiagen column tha
46. uted to a working or use concentration that will consistently give a target C of approximately 25 0 Dilute the stock RNA with sterile RNase free water to the suggested working dilution as described in the product insert sheet that is provided with the transcribed RNA Run the diluted RNA on the rRT PCR assay to determine if the positive NVSL AVPRO1510 02 Testing Protocol Page 17 of 36 Real Time RT PCR Test for Detection of Avian Influenza Virus control has a Cy that is within the acceptable range section 6 2 Positive control transcribed RNA can be requested using the reagent codes listed below Reagent request form 4 9 can be accessed through the NVSL website http www aphis usda gov vs nvsl Home sitemap htm Reagent Code Product Name 200 ADV APMV 1 Transcribed RNA 201 ADV AIV H7 Transcribed RNA 202 ADV AIV H5 Transcribed RNA 203 ADV AIV Matrix Transcribed RNA 3 4 9 Dilute RNase inhibitor to 13 3 units pl with RNase fr water 3 5 Preparation of samples Perform all procedures with potentially live agents in an approved Class II BSC with HEPA filtration Always wear protective clothing safety glasses and gloves when handling potentially infected tissues or live virus Pooled oropharyngeal tracheal swabs 5 swabs tube are the specimen of choice for the PCR procedure and should be extracted using either the Qiagen or Ambion RNA extraction procedure Ap
Download Pdf Manuals
Related Search