G01085 - gerbion
Contents
1. 16 Data Analysis The Chlamydophila pneumoniae specific amplification is measured in the Cy 5 channel the Mycoplasma pneumoniae specific amplification in the ROX channel and the Adenovirus specific amplification in the FAM channel The amplification of the Control DNA K6 is measured in the VIC HEX JOE TET channel Following results can occur A signal in the FAM channel is detected The result is positive the sample contains Adenovirus DNA In this case detection of a signal of the Control DNA K6 in the VICS HEX JOE V TET channel is inessential as high concentrations of virus DNA may reduce or completely inhibit amplification of the Control DNA K6 A signal in the ROX channel is detected The result is positive the sample contains Mycoplasma pneumoniae DNA In this case detection of a signal of the Control DNA K6 in the VICS HEX JOE V TET channel is inessential as high concentrations of bacterial DNA may reduce or completely inhibit amplification of the Control DNA K6 A signal in the CY 5 channel is detected The result is positive the sample contains Chlamydophila pneumoniae DNA In this case detection of a signal of the Control DNA K6 in the VICS HEX JOE V TET channel is inessential as high concentrations of bacterial DNA may reduce or completely inhibit amplification of the Control DNA K6 No signal in the FAM ROX and Cy 5 channel but a signal in the VIC HEX JOE TET channel is detected Th2. ukEx Collection 500 NukEx Collection Tubes for use with GO6008 Tubes NukEx Spin Columns ukEx Universal Diluent for samples for real time RT PCR G01014 Dilution Buffer ukEx Pestle 1 5 100 disposable PBTP pestles for use in 1 5 GO6006 ml ml reaction tubes Individually packed DNase free RNase free non pyrogenic ukEx TS Shredding material aliquoted in 1 5 or 2 0 ml G06007 1 5 safe lock tubes or 2 0 ml screw cap tubes for GO6005 2 0 the manual or automated preparation of G06005 2 0 sc samples such as tissue or insects Proteinase K Proteinase K from Tritirachium album 100 mg GO7001 respiraDNA Instruction Manual Version 1 2 23 02 2015
3. DNA extraction see Control DNA page 7 In this case prepare the Master Mix on ice or in a cooling block 2 to 8 C according to Table 2 respiraDNA Instruction Manual Version 1 2 23 02 2015 The Master Mix contains all of the components needed for PCR except the sample Prepare a volume of Master Mix for at least one sample more than required in order to compensate for pipetting inaccuracy Table 2 Preparation of the Master Mix Control DNA K6 was added during DNA extraction Reaction Volume Master Mix Volume 16 0 ul Reaction Mix K1 16 0 ul x N 1 0 0 ul Control DNA K6 0 0 ul x N 1 Protocol B The Control DNA K6 is used for the control of the real time PCR only see Control DNA page 7 In this case prepare the Master Mix on ice or in a cooling block 2 to 8 C according to Table 3 The Master Mix contains all of the components needed for PCR except the sample Prepare a volume of Master Mix for at least one sample more than required in order to compensate for pipetting inaccuracy Table 3 Preparation of the Master Mix Control DNA K6 is added directly to the Master Mix Reaction Volume Master Mix Volume 16 0 ul Reaction Mix K1 16 0 ul x N 1 0 5 ul Control DNA K6 0 5 pl x N 1 The increase in volume caused by adding the Control DNA K6 is not taken into account when preparing the PCR assay The sensitivity of the detection system is not impaired Protoco
4. A of Chlamydophila pneumonia Mycoplasma pneumonia a clinical specimens e g throat swabs n nd Adenovirus isolated or released from asal swabs bronchial lavage The detection of the amplification is carried out in real time via hybridization and subsequent hydrolysis of the pathogen specific fluorescent probes The respiraDNA Instruction Manual Version 1 2 23 02 2015 fluorescences are measured in the FAM Adenovirus ROX Mycoplasma pneumoniae and Cy 5 channels Chlamydophila pneumoniae Furthermore the respiraDNA real time PCR Kit contains a Control DNA K6 which is detected in a heterologous amplification system Added during DNA extraction the Control DNA K6 allows not only for the detection of PCR inhibition but also detects possible mistakes during DNA extraction This greatly reduces the risk of false negative results The amplification of the Control DNA K6 is measured in the VIC HEX JOE TET channel 10 Equipment and Reagents to be Supplied by User DNA isolation kit e g NukEx Pure RNA DNA gerbion Cat No 605004 or NukEx PLUS 2 0 Nucleic Acid Release Reagent gerbion Cat No 605016 Sterile microtubes Pipets adjustable volume Sterile pipet tips with filter Table centrifuge Vortexer real time PCR instrument Optical PCR reaction tubes with lid Optional Liquid handling system for automation 11 Important Notes The respiraDNA real time PCR must be performed by qualified personnel
5. I8 Other PIOQNCEo susiana naai a E A A GREER 18 respiraDNA Instruction Manual Version 1 2 23 02 2015 1 Components The reagents supplied are sufficient for 32 or 96 reactions respectively Table 1 Components of the respiraDNA real time PCR Kit Label Lid Colour Content 32 96 K1 Reaction Mix yellow 1x512 ul 2 x 768 u K2 Positive Control 1 ed 1x50yu 1x100u Chlamydophila K3 Positive Control 2 ed 1x50yu 1x100u Mycoplasma K4 Positive Control 3 ed 1x50yu 1x100u Adeno K5 Negative Control green 1x50yu 1x100u K6 Control DNA ed 1x160 ul 2x240u 2 Abbreviations PCR Polymerase Chain Reaction DNA Deoxyribonucleic acid 3 Transport and Storage The respiraDNA real time PCR Kit is sh stored at 18 C in the dark immediate Do not use reagents after the date of initial usage reagents are stable fo sensitivity the reagents should not be ipped on dry ice All components must be ly after receipt expiry printed on the package After up to six months To avoid a loss of thawed and frozen more than two times If necessary aliquot kit components K1 K2 K3 K4 and K6 4 ntended Use The respiraDNA real time PCR Kit is a DNA of Adenovirus Mycoplasm pneumoniae in clinical specimens e screening assay for the detection of the a pneumoniae and Chlamydophila g throat swabs nasal swabs bronchial lavage using real time PCR microplate systems resp iraDNA Instruction Manual V
6. components are stored as described in Transport and Storage page 3 Detection of a fluorescence signal in the FAM ROX or Cy 5 channel of the Negative Control K5 Contamination during preparation of the PCR Repeat the real time PCR in replicates If the result is negative in the repetition the contamination occurred when the samples were pipetted into the optical PCR reaction tubes Make sure to pipet the Positive Controls K2 K3 K4 last and close the optical PCR reaction tube immediately after adding the sample If the same result occurs one or more of the kit components might be contaminated Make sure that work space and instruments are decontaminated regularly Use a new kit and repeat the real time PCR respiraDNA Instruction Manual Version 1 2 25 02 2015 18 Other Products A number of products related to real time PCR and nucleic acid isolation is available from gerbion GmbH amp Co KG More information as well as the complete Product Catalogue is available on www gerbion com 18 Product Description Cat No NukEx Pure Spin column based kit for the isolation of GO5004 50 RNA DNA RNA and DNA from a variety of sample G05004 200 matrices For 50 or 200 extractions NukEx PLUS 2 0 Reagent for the enzymatic release of nucleic GO5016 acids from swabs and cell culture suspensions Very fast and convinient protocol Including NukEx Stop for chemical inactivation
7. d connection with asthma is suspected Mycoplasma pneumoniae does usual it is highly contagious and is transn mainly at risk Adenovirus Adenoviruses mainly cau the Serotype numerous keratoconjunctivitis epid Respirato Immuno suppressed pati respiratory distress synd Adenoviruses vary from contact feacal orally and asympton gastro intestinal tract Spreading of the 9 sei rom rarely by Principle of the Test The respiraDNA real time PCR Kit contains specific pri S otitis med y not occur nitted by d nfections of the resp other diseases can be cau emica cystitis rhinitis y symptoms range from mild flu to acu ents are prone to severe complicati e Although the epidemiologica type to type all types natic infections of the palatine and pha all bacteriu m which causes the disease of atypi i cal bacterial pneumonia ia and other symptoms can on It is also associated with al nervous system the liver Furthermore a syndromes in healthy persons however oplet infection Children are iratory system Dependent on sed such as gastroenteritis pharyngitis and diarrhoea te bronchitis and pneumonia ons such as acute characteristics of e transmitted by direct types cause persistent yngeal tonsils and the r over months or years a ne water Son virus can occu mers and probes labelled with a fluorescent dye for the analysis of the DN
8. d on www gerbion com Important In addition to the samples always run a water control in your extraction possible contaminations during DNA extraction will be detectable Treat this water control analogous to a sample Please note the chapter Control DNA on page 7 If the real time PCR is not performed immediately store extracted DNA according to the instructions given by the DNA extraction kit s manufacturer Further information about DNA isolation is to be found in the extraction kit manual or from the extraction kit manufacturer s technical service 14 Control DNA K6 The respiraDNA real time PCR Kit contains a Control DNA K6 which allows the user to control the DNA isolation procedure and to check for possible real time PCR inhibition Control DNA K6 used as Extraction Control respiraDNA Control DNA K6 is added prior to the DNA extraction To this end multiply the buffer volume needed per extraction with the number of samples including at least one water control N plus 1 to compensate for respiraDNA Instruction Manual Version 1 2 23 02 2015 inaccuracies in pipetting N 1 Add 5 ul Control DNA K6 per extraction 5 pl x N 1 Mix well Perform the DNA isolation according to the manufacturer s instructions If the extraction protocol includes an incubation step of the sample in the first buffer the Control DNA K6 is to be added to each sample individually after incubation The Contro
9. e of expiry for one or more kit printed on the kit label If necessary use a new kit and components or kit expired make sure kit components are stored as described in Transport and Storage page 3 real time PCR conditions do Check the real time PCR conditions page 8 not comply with the protocol real time PCR inhibited Make sure that you use an appropriate isolation method see Isolation of DNA page 7 and follow the manufacturers instructions Make sure that the ethanol containing wash buffer of the isolation kit has been completely removed An additional centrifugation step at high speed is recommended before elution of the DNA Dilute NukEx PLUS 2 0 respiraDNA Instruction Manual Version 1 2 23 02 2015 17 lysates 1 3 in gH20 or NukEx Universal Dilution Buffer gerbion Cat No GO1014 Alternatively purify the lysates with e g NukEx Pure RNA DNA Kit gerbion Cat No 605004 DNA loss during isolation process In case the Control DNA K6 was added during extraction the lack of an amplification signal can indicate that the DNA isolation was not successful Make sure that you use an appropriate isolation method commercial kits are recommended and stick to the manufacturer s protocol Incorrect storage conditions for one or more components or kit expired Check the storage conditions and the date of expiry printed on the kit label If necessary use a new kit and make sure kit
10. e result is negative the sample does neither contain Chlamydophila pneumoniae DNA nor Mycoplasma pneumonia DNA nor Adenovirus DNA respiraDNA Instruction Manual Version 1 2 23 02 2015 14 The signal of the Control DNA K6 excludes the possibilities of DNA isolation failure in case the Control DNA K6 is being used as an Extraction Control and or real time PCR inhibition If the C value of a sample differs significantly from the C value of the water control a partial inhibition occured which can lead to negative results in weak positive samples see Troubleshooting page 16 Neither in the FAM ROX Cy 5 nor in the VIC HEX JOE TET channel a signal is detected A diagnostic statement cannot be made The DNA isolation was not successful or an inhibition of the PCR has occurred In case the Control DNA K6 was added during DNA isolation and not directly to the PCR Master Mix the Negative Control K5 is negative in both channels respiraDNA Instruction Manual Version 1 2 23 02 2015 15 Figure 1 and Figure 2 show examples for positive and negative real time PCR results positive sample negative sample e e 9 e ce eee aa 2 4 95 8 0 2 14 16 19 20 22 24 120 1 30 32 34 36 3 4 42 4 Cycles Figure 1 The positive sample shows bacteria specific amplification in the FAM channel whereas no fluorescence signal is detected in the negative sample Fluoresce
11. ersion 1 2 23 02 2015 5 Sample Material Starting material for the assay is DNA isolated or released from clinical specimens e g throat swabs nasal swabs bronchial lavage 6 Quality Control In accordance with gerbion s ISO certified Quality Management System each lot of the respiraDNA real time PCR Kit is tested against predetermined specifications to ensure consistent product quality 7 Product Warranty gerbion guarantees the performance of all products when used according to the instructions given in the Instruction Manual The purchaser must determine the suitability of the product for its particular use Should any product fail to perform satisfactorily due to any reason other than misuse gerbion will replace it free of charge or refund the price We reserve the right to change alter or modify any product to enhance its performance and design 8 Introduction respiraDNA is a multiplex real time PCR for the detection of causative agents of respiratory diseases The respiraDNA real time PCR is designated for pathogens with a DNA genome Chlamydophila pneumoniae Mycoplasma pneumoniae and Adenovirus In combination with NukEx PLUS 2 0 gerbion Cat No GO5016 for the preparation of throat swabs and nasal swabs respiraDNA real time PCR allows for fast efficient and cost effective diagnostics Chlamydophila pneumoniae Chlamydophila pneumoniae formerly Chlamydia pneumoniae are obligate intracellular bacteria which are mainly
12. ger ar Instruction Manual respiraDNA real time PCR Kit For research use only For the in vitro detection of the DNA of Adenovirus Mycoplasma pneumoniae Chlamydophila pneumoniae in clinical specimens G01085 32 G01085 96 32 96 18 C gerbion gmbH amp Co KG Remsstr 1 70806 Kornwestheim Germany phone 49 7154 806 200 fax 49 7154 806 20 29 e mail info gerbion com www gerbion com E gt respiraDNA Instruction Manual Version 1 2 23 02 2015 Index 1 COrmponelitez oen e Uem n tonat e ndum RR 5 2 AbDreVIatiOfiS cotto tasa ee Mie tet a e detect 5 3 Trarisportiand Stordge see met eo a E EEEE NAE AR 3 4 Intended USE ia citera A a RA INEN coll AE NE INE 5 5 Sample Material octo Re AIRTRAN E CA RR OEE A 4 6 Quality CODO tests tette rte tote te bedeutend 4 7 Product Warranty 8 pigeja iu eio pe 4 9 Priaciple of the Test oett ut dee titan 5 10 Equipment and Reagents to be Supplied by User 6 os important Noles soccer c e etae e coe cu 6 I2 General Precautions u c tiir nae ER ANEREN 6 T5 ISOLATION ot DNA ettet e eet ito en ete qeu 7 T4 Control DNA K6 stt eet e a eio ee e trei 7 I5 Realtime POR tetto eet atender icon debet 8 151 Important Points Before Starting ttt 8 152 PROCECUIEC E 8 15 5 Instr ment Settirigs ett cette ore ette 10 T6 ata AnalysiS ettet ve Da b teet en dnd Pte ttt 15 17 ThoublesHaotlg sestertia dta 16
13. l A and B real time PCR set up Put the number of optical PCR reaction tubes needed into the cooling block Pipet 16 ul of the Master Mix into each optical PCR reaction tube Add 4 ul of the eluates from the DNA isolation including the eluate of the water control or the crude NukEx PLUS 2 0 lysates the Positive Controls K2 K5 K4 and the Negative Control K5 to the corresponding optical PCR reaction tube Table 4 Close the optical PCR reaction tubes immediately after filling in order to reduce the risk of contamination respiraDNA Instruction Manual Version 1 2 23 02 2015 10 Table 4 Preparation of the real time PCR Component Volume Master Mix 160u Sample 4 0 u Total Volume 20 0 u 15 3 Instrument Settings For the real time PCR use the thermal profile shown in Table 5 Table 5 real time PCR thermal profile Discription Time Temperature Number of Cycles QUK Initial Denaturation 5 15min 95 C 1 Amplification of DNA Denaturation 10 sec 95 C 45 Annealing and Extension 40 sec 60 C Aquisition at the end of this step When using crude NukEx PLUS 2 0 lysates that have not been heat inactivated or purified the initial denaturation has to be increased to 15 min For purified DNA samples 5 min denaturation is sufficient Samples can be tested for pathogens with a RNA genome in the same PCR run eg with the respiraRNA real time RT PCR Kit when a reverse transcripti
14. l DNA K6 must not be added to the sample material directly Control DNA K6 used as Internal Control of the real time PCR If crude NukEx PLUS 2 0 lysates are being used or control of the DNA extraction is not desired the Control DNA K6 can be used as Internal Control of the real time PCR only To that end the Control DNA K6 is to be added directly to the real time PCR Master Mix 15 Realtime PCR 15 1 Important Points Before Starting Please pay attention to the Important Notes on page 6 Before setting up the real time PCR familiarise yourself with the real time PCR instrument and read the user manual supplied with the instrument The programming of the thermal profile should take place before the PCR set up In every PCR run at least one of each Positive Control K2 K3 K4 and one Negative Control K5 should be included Before each use all reagents should be thawed completely at room temperature thouroughly mixed do NOT vortex the Reaction Mix K1 but mix by pipetting up and down repeatedly and centrifuged very briefly Then place all reagents on ice or on a cooling block 2 to 8 C 15 2 Procedure If the Control DNA K6 is used to control both the real time PCR and the DNA isolation procedure please follow protocol A If the Control DNA K6 is solely used to detect possible inhibition failure of the real time PCR please follow protocol B Protocol A The Control DNA K6 was added during
15. nce dR 2 4 0 89 0 12 1 19 1 2 22 24 2 28 30 32 24 36 39 4 42 4 Cycles Figure 2 The positive sample as well as the negative sample show a signal in the Control DNA specific VICS HEX JOE V TET channel The amplification signal of the Control DNA K6 in the negative sample shows that the missing signal in the bacteria specific FAM channel is not due to PCR inhibition or failure of DNA isolation but that the sample is a true negative respiraDNA Instruction Manual Version 1 2 23 02 2015 17 Troubleshooting The following troubleshooting guide is included to help you with possible problems that may arise when performing a real time PCR If you have further questions please do not hesitate to contact our scientists on info gerbion com The selected channel for Select the FAM channel for analysis of the Adenovirus analysis does not comply specific amplification the ROX channel for the with the protocol Mycoplasma pneumoniae specific amplification the Cy 5 channel for the Chlamydophila pneumoniae specific amplification and the VIC HEX JOE TET channel for the amplification of the Control DNA K6 Incorrect configuration of Check your work steps and compare with Procedure the real time PCR on page 8 The programming of the Compare the thermal profile with the protocol Table thermal profile is incorrect 5 page 10 Incorrect storage conditions Check the storage conditions and the dat
16. on step is run prior to the amplification cycles The thermal profile has to be programed according to Table 6 Important Crude NukEx PLUS 2 0 lysates must be heat inactivated prior to adding them to the real time RT PCR mix when performing a reverse transcriptase step prior to the initial denaturation respiraDNA Instruction Manual Version 1 2 23 02 2015 1l Table 6 real time RT PCR thermal profile Reverse Transcription 10 min 45 C 1 Initial Denaturation 5 min 95 C 1 Amplification of DNA Denaturation 10 sec 95 C 45 Annealing and Extension 40 sec 60 C Aquisition at the end of this step Dependent on the real time instrument used further instrument settings have to be adjusted according to Table 7 respiraDNA Instruction Manual Version 1 2 23 02 2015 12 Table 7 Overview of the instrument settings required for the respiraDNA real time PCR Adenovirus 485 555 LightCycler 48011 M pneumoniae 338 619 ares i PEU EAE Control DNA 615 670 required C pneumoniae 523 568 Adenovirus FAM Gain 8 Stratagene Mx5000P M pneumoniae ROX Gain 1 Reference Dye DR ControlDNA HEX Gan None C pneumoniae Cy5 Gain 4 Adenovirus FAM ABI 7500 M pneumoniae ROX Option Reference Dye ROX Control DNA JOE NS C pneumoniae Cy5 Adenovirus Green ipaa s 0 M pneumoniae Orange Rotor Gene 6000 Control DNA Yellow C pneumoniae Red respiraDNA Instruction Manual Version 1 2 23 02 2015 13
17. only Good Laboratory Practice GLP has to be applied All samples must be regarded as potentially infectious material and all equipment used has to be treated as potentially contaminated 12 General Precautions Stick to the protocol described in the Instruction Manual Set up different laboratory areas for the preparation of samples and for the set up of the PCR in order to avoid contaminations Pipettes tubes and other materials must not circulate between those different laboratory areas Always use filter tips respiraDNA Instruction Manual Version 1 2 23 02 2015 Regulary decontaminate equipment and benches with ethanol free decontaminant Do not combine respiraDNA real time PCR Kit components of different lot numbers 13 Isolation of DNA The respiraDNA real time PCR is suitable for the detection of Chlamydophila pneumoniae Mycoplasma pneumonia and Adenovirus DNA isolated or released from clinical specimens e g throat swabs nasal swabs bronchial lavage with appropriate isolation methods Commercial kits for DNA isolation are recommended e g NukEx Pure RNA DNA gerbion Cat No 605004 Alternatively DNA can be released from throat or nasal swabs with NukEx PLUS 2 0 Nucleic Acid Release Reagent gerbion Cat No G05016 This is the fastest and most convenient method for the release of nucleic acid from swabs because column based purification of the DNA can be omitted More information can be foun
18. transmitted by droplet infection C pneumoniae is a common cause of pneumonia around the world it is typically acquired by otherwise healthy people and is a form of community acquired pneumonia The infestation of the human population is very high Antibodies against Chlamydophila pneumoniae can be found in around 70 to 8096 of the 60 year olds and in around 3096 of ten year old school children Because its treatment and diagnosis are different from historically recognized causes such as Streptococcus pneumoniae pneumonia caused by C pneumoniae is categorized as an atypical pneumonia Most infections remain without or only mild symptoms Bronchitis sinusitis chronical obstructive respiraDNA Instruction Manual Version 1 2 23 02 2015 respiratory diseases can occur Typical early symptoms of a Chlamydophila pneumoniae infection are dry mucous to 6 weeks after the primary in tenosynovitis might occur In immuno suppressed persons such patients and in elder Mycoplasma pneumoniae membranes of mouth nose and eyes 4 X ection post infectious arthritis and as cancer HIV or organ transplant y people severe complications can lead to fatal outcome Mycoplasma pneumoniae is a very sm mycoplasma pneumonia a form Tracheobronchitis laryngitis meningiti be caused by Mycoplasma pneumoniae infecti disorders of the haematopoietic system the cent iovascular and the pancreas and with car
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