Bordetella pertussis B parapertussis B - bio
Contents
1. When calculating the volume of the reaction mixture take into account controls negative control of extraction positive and negative controls of amplification Refer to Table 1 for calculated reaction volumes Table 1 Reaction mixture preparation scheme NO a a Reagent volume for Volume of reagents for indicated number of reactions one reaction pil 10 00 5 00 0 50 i sea i ui PCR mix 2 FRT ul TagF Polymerase pi 2 60 30 3 0 4 80 40 4 0 6 100 50 5 0 8 120 60 6 0 10 140 70 7 0 12 160 80 8 0 14 180 90 9 0 16 200 100 10 0 Number of samples including DNA extraction and amplification controls N 4 Mix the tube with the prepared mixture and vortex it briefly Transfer 15 ul of the prepared mixture to prepared tubes Add 10 ul of DNA obtained after nucleic acid extraction from clinical or control samples Carry out the control amplification reactions NCA Add 10 pl of TE buffer to the tube labeled NCA Negative control of amplification C Add 10 ul of Positive Control DNA Bordetella spp to the tube labeled C Positive control of amplification IC Add 10 ul of Positive Control ICto the tube labeled CS Positive control of IC amplification C Add 10 pl of a sample extracted from the Negative Control to the tube labeled C Negative Control of Extraction VER 21 03 2013 It is recommended to sediment drops from walls of tubes by short vortex 1 3 s befo2. TaqF Polymerase 0 06 ml Pos Control DNA Bordetella spp C 2 x 0 1 ml Positive Control IC 2 x 0 1 ml TE buffer 2 x 0 5 ml Negative Control C 1 2 ml Internal Control IC 2 x 0 6 ml Contains reagents for 100 reactions including controls must be used in the extraction procedure as Negative Control of Extraction add 10 ul of Internal Control during the DNA extraction procedure directly to the sample lysis mixture MATERIALS REQUIRED BUT NOT PROVIDED DNA extraction kit Disposable powder free gloves and laboratory coat Automatic adjustable pipettes from 5 to 20 ul and from 20 to 200 ul Disposable tips with aerosol barriers 100 or 200 ul in tube racks Tube racks Vortex mixer desktop centrifuge PCR box Real Time PCR instrument Disposable polypropylene microtubes for PCR or PCR plate Refrigerator for 2 8 C Deep freezer for lt 16 C VER 21 03 2013 STORAGE INSTRUCTIONS All components of theBordetella pertussis B parapertussis B bronchiseptica Real TM PCR kit except for PCR mix 1 FRT PCR mix 2 FRT and TaqF Polymerase are to be stored at 2 8 C when not in use All components of the Bordetella pertussis B parapertussis B bronchiseptica Real TM PCR kit are stable until the expiration date on the label The shelf life of reagents before and after the first use is the same unless otherwise stated PCR mix 1 PCR mix 2 FRT and TaqF Polymerase are to be stored at lt 16 C AN PCR
3. PCR fluorescent curve should have typical exponential increase of fluorescent signal In such samples Ct for internal control IC FAM can have any value or be absent in case of high pathogen load in a sample B parapertussis DNA is detected in a sample if the Ct value of a sample specified in the table for the HEX channel does not exceed 25 and Ct values for the ROX and Cy5 channels are absent In such samples Ct for internal control IC FAM can have any value or be absent in case of high pathogen load in a sample Bordetella spp B pertussis or B parapertussis or B bronchiseptica DNA is detected if Ct value of a sample specified in the table for the JOE Yellow channel exceed 25 Ct values for the ROX and Cy5 channels are absent Ct for internal control IC FAM does not exceed 29 That can take place if the quantity of extracted DNA is insufficient For species differentiation repeat sampling and nucleic acid extraction steps Results should be interpreted as equivocal if a repeated PCR analysis demonstrates absent Ct value in the HEX channel while Ct values in the ROX or Cy5 and FAM channels do not exceed 29 Repeat of sampling is advised Results should be interpreted as invalid if the Ct values of a sample specified in the table for the ROX and Cy5 channels have not been detected absent for the HEX channel is absent or exceed 25 and for internal control IC FAM is either absent or exceed 29 In this case repe
4. of the statedcausative agents The specificity of this kit was confirmed by investigation of the following reference strains Streptococcusspp Moraxella catarrhalis Staphilococcus aureus Staphilococcus saprophiticus Haemophilus influenzae Proteus mirabilis Klebsiella pneumoniae Pseudomonas aeruginosa Mycobacteria tuberculosis 27294 105 Neisseria flava Neisseria sicca Neisseria mucosa E coli ATCC NCTC 01577 27u7 Enterococcus faecalis Mycoplasma pneumoniae Chlamydophila pneumoniae Legionella pneumophila Shigella flexneri Shigella sonnei Salmonella Enteritidis Yersinia enterocollitica as well as human genomic DNA TROUBLESHOOTING Results of analysis are not taken into account in the following cases 1 If Ct value of positive control DNA Bordetella spp C in the appropriate channel is absent or exceeds boundary value amplification stage is to be repeated for all clinical samples interpreted as negative 2 If Ct value of negative control of extraction C in the JOE HEX Yellow ROX Orange andCy5 Red channels and or negative control of amplification NCA in any fluorescent channel is detected test analysis must be repeated from the extraction stage and measures to detect and eliminate the source of possible contamination must be taken 3 If a positive result the fluorescence curve crosses the threshold line is detected for a sample that has a fluorescence curve without the typical exponential growth phase the curve
5. the area of reliable increment of fluorescence and Ct value in the results grid for a particular channel is less than the indicated boundary value negative in case of absence of typically S shaped curve not crossing the threshold line does not have Ct or Ct exceeds the indicated boundary value The results of analysis are considered reliable only if the results obtained for positive and negative controls of amplification and negative control of extraction are correct see Table 2 Table 2 Results for controls Value of threshold cycle Ct Control Stage for control NCE DNA extraction lt 30 Neg Neg Neg NCA Amplification Neg Neg Neg Neg IC Amplification lt 27 Neg Neg Neg C Amplification Neg lt 25 lt 27 lt 26 VER 21 03 2013 Principle of interpretation of results Interpretation of results of PCR analysis for detection and differentiation of pathogens that cause whooping cough Bordetella pertussis parapertussis Bordetellaparapertussis and Bordetellabronchisepticainfection Bordetellabronchiseptica is based on amplification results in accordance with Table 3 Table 3 Interpretation of results for PCR analysis Variants Interpretation Value of threshold cycle Ct absentor absentor 3 1 absent absent Invalid gt 29 gt 25 B pertussis 2 lt 29 absent absent absent B parapertussis B bronchiseptica NOT detected lt i 3 ea oF lt 35 lt 35 absent B pert
6. _lSacace BIOTECHNOLOGIES w IVD For in Vitro Diagnostic Use CE Bordetella pertussis B parapertussis B bronchiseptica Real TM Handbook Real Time PCR test for detection and differentiation of Bordetella pertussis Bordetella parapertussis and Bordetella bronchiseptica REF B84 100FRT amp 100 Sacace Bordetella pertussis B parapertussis B bronchiseptica Real TM VER 21 03 2013 NAME Bordetella pertussis B parapertussis B bronchiseptica Real TM INTRODUCTION Bordetella is a genus of small 0 2 0 7 um Gram negativecoccobacilli of the phylum proteobacteria Three species are human respiratorypathogens B pertussis B parapertussis and B bronchiseptica Bordetella pertussis is an obligate human pathogen and is the causative agent of whooping cough pertussis Bordetellaparapertussis causes a milder form of disease in humans and also causes respiratory infections in sheep Bordetellabronchisepticahas the broadest host range causing disease in many mammalian species but kennel cough in dogs and atrophic rhinitis in which infected piglets develop deformed nasal passages have the biggest economic impact Humans however arerarelyinfectedwiththisorganism Wheninfectionsinhumansoccur theyar eoftenacquiredthroughanimalcontactandtypicallyinvolveimmunocompromised patients Thespectrumofimmunocompromisedpatientswhohavedeveloped B bronchiseptica infectionincludespatientswithmalignanciessuchasHodgkin s disease
7. at nucleic acid extraction step for the required clinical sample Results of analysis are not taken into account in the following cases If Ct value of positive control DNA Bordetella spp C in the appropriate channel is absent or exceeds boundary value see Table 3 repeat amplification for all negative clinical samples The samples with negative result in all channels should be analyzed once more starting from the nucleic acid extraction stage If negative result is obtained in the second run repeat collection of the clinical material Negative result is normally detected only for the Negative Control of amplification NCA If the Ct value for the Positive Control of amplification C is absent or exceeds the boundary Ct value in the appropriate channel repeat amplification for all negative clinical samples If the Ct value is present for the Negative Control of extraction C and or Negative Control of amplification NCA in the channel for detection of any gene target HEX ROX andCy5 repeat the analysis for all samples in which the specific gene was detected starting from the nucleic acid extraction stage to rule out possible contamination VER 21 03 2013 SPECIFICATIONS Sensitivity The analytical sensitivity of Bordetella multiplex Real TMPCR kit for the stated causative agents is no less than 5x10 copies per 1 ml of sample Specificity Bordetella multiplex Real TMPCR kit makes it possible to detect DNA specific regions
8. chroniclymphocytic leukemia cardiacand bone marrow transplantation patients patients with AIDS INTENDED USE Bordetella pertussis B parapertussis B bronchiseptica Real TMPCR kit is an in vitro nucleic acid amplification test for detection and differentiation of pathogens that cause whooping cough Bordetella pertussis parapertussis Bordetellaparapertussis and Bordetellabronchiseptica infection Bordetellabronchiseptica in the clinical materials nasal and oropharyngeal swabs and culture of microorganisms by using real time hybridization fluorescence detection VER 21 03 2013 PRINCIPLE OF ASSAY Bordetella determination by the polymerase chain reaction PCR with hybridization fluorescent detection includes three stages DNA extraction from clinical samples PCR amplification of pathogen genome specific region and real time hybridization fluorescent detection DNA is extracted from samples in presence of Internal Control IC which allows to monitor the analysisof each sample In real time PCR the amplified product is detected using fluorescent dyes These dyes are linked to oligonucleotide probes which bind specifically to the amplified product during thermocycling The real time monitoring of the fluorescence intensities during the real time PCR allows the detection of PCRproduct without re opening the reaction tubes after the PCR run During the amplification stage four simultaneous reactions take place amplification of the conse
9. ification EN375 Strict compliance with the user manual is required for optimal PCR results Attention should be paid to expiration dates printed on the box and labels of all components Do not use a kit after its expiration date SAMPLE COLLECTION AND TRANSPORT Bordetella pertussis B parapertussis B bronchiseptica Real TM PCR kitis intended for the analysis ofDNA extracted from Posterior nasopharynx swabs Throat swabs and anterior nasal swabs have unacceptably low rates of DNA recovery and should not be used for pertussis diagnosis The swab tips may be polyester such as Dacron rayon or nylon flocked Cotton tipped or calcium alginate swabs are not acceptable as residues present in these materials inhibit PCR assays When the material is obtained place the working part of the swab in a sterile disposable tube with 500 ul of transport medium for storage and transportation of respiratory swabs Break off the terminal part of the probe to allow tight closing of the tube cup Close the tube with the solution and the working part of the probe and mark it Oropharyngeal swabs are taken with a probe with a dry viscose swab Take swabs by rotating the probe over the surface of tonsils palatine arches and the posterior wall of the pharynx When the material is obtained place the working part of the swab in a sterile disposable tube with 500 ul of transport medium for storage and transportation of respiratory swabs Break off the terminal par
10. is linear this may suggest incorrect setting of the threshold line or incorrect calculation of baseline parameters Such a result should not be considered as positive If you have any further questions or if you encounter problems please contact our Authorized representative in the European Community VER 21 03 2013 Sacace Bordetella pertussis B parapertussis B bronchiseptica Real TM VER 21 03 2013 KEY TO SYMBOLS USED el Be 2 List Number Lot Number For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date SaCycler is a registered trademark of Sacace Biotechnologies CFX and iQ5 are trademarks of Bio Rad Laboratories Rotor Gene is a registered trademark of Qiagen MX3005P are trademarks of Agilent Technologies ABI is trademarks of Applied Biosystems SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl VER NCA NCE C Caution Contains sufficient for lt n gt tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification Internal Control via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com VER 21 03 2013
11. mix 1is to be kept away from light STABILITY Bordetella pertussis B parapertussis B bronchiseptica Real TM Test is stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Repeated thawing and freezing of these reagents should be avoided as this may reduce the sensitivity Components stored under conditions other than thoseonesstated on the labels may not perform properly and may adversely affect the assay results QUALITY CONTROL In accordance with Sacace s ISO 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only The user should always pay attention to the following e Use sterile pipette tips with aerosol barriers and use new tip for every procedure e Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughl
12. ple during the last amplification cycle VER 21 03 2013 DATA ANALYSIS The results are analyzed by the software oftheused real time PCR Instrument Interpretation of the results is performed by the crossing or not of the fluorescence curve with the adjusted threshold line for each single fluorescent channel employed It defines presence or absence of threshold cycle Ct for a certain DNA sample in the according graph of the resultant table Principle of interpretation of results PCR curves of accumulatedfluorescent signal are analyzed in four channels Detection channel Detection of Identification Identification of Result IC detection pertussis toxin of Bordetella Bordetella gene ptxA pertussis bronchiseptica in the JOE HEX Yellow channel signal indicating amplification of pertussis toxin gene fragment present in genomes of Bordetella pertussis Bordetella parapertussis and Bordetella bronchiseptica is registered in the ROX Orangechannel signal indicating amplification of genome s specific region of Bordetella pertussis is registered in the Cy5 Red channel signal indicating amplification of genome s specific region of Bordetella bronchiseptica is registered inthe FAM Green channel signal indicating amplification of Internal Controlis registered The result of amplification is considered to be positive if the real time PCR fluorescent curve is typically S shaped crosses threshold line in
13. re inserting them in the thermalcycler Amplification 1 Create a temperature profile on your instrument as follows Rotor type instruments Plate type instruments Step Temperature C Time Cycles Temperature C Time Cycles 1 95 15 min 1 95 15 min 1 95 10s 95 10s 2 60 20s 10 60 25s 10 72 10s 72 25s 95 10s 95 10s 20s 30s 3 60 Fluorescence 35 60 Fluorescence 35 detection detection 72 10s 72 25s T For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example iQ5 BioRad Mx3005P Agilent ABI 7500 Applied SmartCycler Cepheid SaCycler 967M Sacace Fluorescent signal is detected in FAM Green JOE Yellow HEX ROX Orange and Cy5 Red channels 2 Place PCR tubes into the PCR instrument 3 Run amplification and signal detection program 4 After measurement start data analysis and interpretation of results INSTRUMENT SETTINGS Rotor type instruments RotorGene 6000 Q Channel Threshold perio E ponent Slope Correct FAM Green 0 1 from 5FI to 10Fl 0 Off JOE Yellow 0 1 from 5FI to 10Fl 5 Off Rox Orange 0 1 from 5FI to 10Fl 5 Off Cy5 Red 0 1 from 5FI to 10Fl 5 Off Plate or modular type instruments SaCycler iQ5 Mx300P ABI 7500 SmartCycler For result analysis set the threshold line at a level corresponding to 10 20 of the maximum fluorescence signal obtained for Pos C sam
14. rvative region of ptxA gene that codes pertussis toxin located in Bordetella pertussis Bordetella parapertussis and Bordetella bronchiseptica genomes identification of specific regions in genomes of Bordetella pertussis and Bordetella bronchiseptica as well as amplification of nucleic acid sequence of the Internal Control IC sample Detection channel Detection of Identification Identification of Result IC detection pertussis toxin of Bordetella Bordetella gene ptxA pertussis bronchiseptica In case of pertussis toxin gene detection JOE HEX Yellow channel the conclusion about presence of Bordetella spp B pertussis B parapertussis or B bronchiseptica is made If results are concurrently positiveboth in JOE HEX Yellow and ROX Orange channels the sample is considered positive for Bordetella pertussis If results are concurrently positive both inJOE HEX Yellow and Cy5 Red channels the sample is considered positive for Bordetella bronchiseptica Presence of Bordetella parapertussis can be concluded in case of pertussis toxin gene detection JOE HEX Yellow channel in the sample negative for Bordetella pertussis and Bordetella bronchiseptica providing that sufficient Bordetella DNA is available VER 21 03 2013 MATERIAL PROVIDED Module No 1 Real Time PCR kit B84 100FRT Bordetella pertussis B parapertussis B bronchiseptica Real TM Real Time amplification PCR mix 1 FRT Bordetella 5 x 0 2 ml PCR mix 2 FRT 0 6 ml
15. t of the probe to allow tight closing of the tube cap Close the tube with the solution and the working part of the probe and mark it Storage of clinical specimens is allowed for 3 days at 2 8 C or during a week at lt 16 Microorganism cultures are resuspended in 1 ml of 0 9 saline or 0 01 Mphosphate buffer pH 7 0 The obtained suspension is used for subsequent DNA extraction Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents DNA ISOLATION The following kits are recommended gt DNA RNA Prep Sacace REF K 2 9 gt Ribo Sorb Sacace REF K 2 1 Please carry out DNA extraction according to the manufacture s instruction DNA is extracted from each clinical sample in the presence of Internal Control 10pl A of IC is added to each sample Transfer 100pl of Negative Control to the tube labeled C VER 21 03 2013 PCR PROTOCOL 1 Prepare the required number of tubes for amplification of DNA from clinical and control samples Thaw the tubes with PCR mix 1 FRTBordetella VortexPCR mix 1 FRTBordetella PCR mix 2 FRT polymerase TaqF and centrifuge briefly 1 2 sec Prepare the required number of tubes or strips including controls for DNA amplification For N reactions add to a new tube 10 N 1 pl ofPCR mix 1 FRT Bordetella 5 0 N 1 pl of PCR mix 2 FRT 0 5 N 1 ul offaqF polymerase
16. ussis DNA absent detected i 35 or lt 35 absent lt 35 B bronchiseptica absent DNA detected lt 35 or A 5 lt 25 absent absent B parapertussis absent DNA detected Bordetella spp DNA detected B pertussis or 6 lt 29 gt 25 absent absent B parapertussis or B bronchiseptica For differentiation repeat sampling and extraction If the result is 7 lt 29 absent lt 35 absent repeated in PCR interpret as equivocal If the result is 8 lt 29 absent absent 35 repeated in PCR interpret as equivocal Bordetella pertussis Bordetellaparapertussis and Bordetellabronchiseptica is not detected in a sampleif the Ct value of a sample specified in the table for the HEX ROX andCy5 channels has not been detected absent i e the PCR florescent curve does not cross the threshold line Herewith Ct value for internal control IC FAM does not exceed 29 Bordetellapertussis DNA is detected in a sample if the Ct value of a sample specified in the table for theHEX and ROX channelsis less or equal to 35 and the PCR fluorescent curve should have typical exponential increase of fluorescent signal In such samples Ct for internal control IC FAM can have any value or be absent in case of high pathogen load in a sample VER 21 03 2013 B bronchiseptica DNA is detected in a sample if the Ct value of a sample specified in the table for the HEX and Cy5channels is less or equal to 35 and the
17. y wash hands afterwards e Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas e Do not use a kit after its expiration date e Dispose of all specimens and unused reagents in accordance with local authorities regulations e Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices e Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant e Avoid sample or reagent contact with the skin eyes and mucous membranes If skin eyes or mucous membranes come into contact rinse immediately with water and seek medical advice immediately e Material Safety Data Sheets MSDS are available on request e Use of this product should be limited to personnel trained in the techniques of DNA amplification e The laboratory process must be one directional it should begin in the Extraction Area and then move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous step was performed Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer VER 21 03 2013 PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnostics Use of this product should be limited to personnel trained in the techniques of DNA ampl
Download Pdf Manuals
Related Search