User manual REALQUALITY RS
Contents
1. BUT NOT PROVIDED 6 1 REAGENTS Reagents for DNA extraction DNAse and RNAse free sterile water REALQUALITY RQ MYCO P STANDARD code RQ 48 ST for quantitative analysis 6 2 INSTRUMENTS Laminar flow cabinet its use is recommended while preparing the amplification mix to avoid contamination it is recommended to use another similar laminar flow cabinet to add the extracted DNA positive controls and or standards Micropipettes range 0 5 10 uL 2 20 uL 10 100 uL 20 200 uL 100 1000 UL Microcentrifuge max 12 14 000 rpm Plate centrifuge optional Real time amplification instrument This kit was standardized on Applied Biosystems 7500 Fast Dx 7300 and StepOnePlus Real time PCR Systems Applied Biosystems The kit can be used on instruments that work with 25 uL of reaction volume and can detect the FAM and JOE fluorophores The JOE fluorophore depending on the instrument can also be detected in CY3 and HEX channels etc For further information on instrument compatibility of the kit please contact AB ANALITICA s technical support 6 3 DISPOSABLES Talc free disposable gloves Disposable sterile filter tips range 0 5 10 uL 2 20 uL 10 100 uL 20 200 uL 100 1000 uL 96 well plates for Real time PCR and optical adhesive film or 0 1 0 2 mL tubes with optical caps 547 48 EN doc 8 Qanauitica www abanaliticait 7 INTRODUCTION Mycoplasma pneumoniae is the most common etiological agents of prim2. 1 DNA EXTRACTION For DNA extraction we recommend to use the QlAamp DNA Mini Kit QIAGEN Hilden Germany For use refer to the manufacturer s instructions Folow for each application use the most appropriate protocol as recommended by the manufacturer This IVD device can be used with DNA extracted with the most common manual and automated extraction methods For further information regarding the compatibility of the device with different extraction methods please contact AB ANALITICA s technical support 11 2 INTERNAL CONTROL This kit includes an internal control IC consisting of recombinant DNA containing part of the B globin gene BG The use of this control is recommended for analysis of acellular samples and provides a means of verifying the extraction procedure as well as detecting inhibition of the amplification reaction For use of the internal control follow to the instructions below The internal control has to be added at the beginning of DNA extraction procedure e g during the lysis step This is important for verifying the extraction procedure properly Do NOT add the control to the untreated sample this may result in degradation of the DNA in contained in the control If possible use the internal control IC in a ratio of 1 6 in respect to the final DNA elution volume For example use 10 uL of internal control if the final elution volume is 60 yl Also refer to the instructions provided by the manufacturer
3. 1354 1985 Schrag SJ et al Pediatr Infect Dis J 19 17 22 2000 Sriram et al Ann Neurol 46 6 14 1999 Tuuminen T et al Clin Diagn Lab Immunol 7 734 738 2000 27 RQ S47 48 EN doc www abanalitica 16 RELATED PRODUCTS REALQUALITY RQ MYCO P STANDARD Quantification standard for Mycoplasma pneumoniae quantified and ready to use This product is in accordance with 98 79 CE Directive Annex III regarding the in vitro medical diagnostic devices CE mark Code Product PKG REALQUALITY RQ MYCO P RQ S46 ST STANDARD 4 x 60 uL 547 48 EN doc Qanauitica 2 8 www abanaliticait ANALITICA ADVANCED BIOMEDICINE www abanalitica it AB ANALITICA srl Via Svizzera 16 35127s PADOVA ITALY Tel 39 049 761698 Fax 39 049 8709510 e mail info abanalitica it
4. 19 RQ S47 48 EN doc 11 7 QUANTITATIVE ANALYSIS AND INTERPRETATION OF RESULTS When the PCR run is finished analyze the amplification results separately for control gene B globin and MYCO P View the analysis graph in logarithmic scale see Figure 2 Set the threshold so you obtain a correlation coefficient R and slope of the standard curve Fig 3 as close as possible to the optimum R 1 00 slope 3 33 Values of R 2 0990 and a slope in the range of 3 75 and 3 10 amplification efficiency 85 110 are acceptable values Delta Rn vs Cycle 1 000 001 1 000 H dd 1 000e 001 1 000 002 Delta Rn SS S 1 0006 003 SN Sy st dp NI NY 4 2501 YN 1 000e 004 H 1 1 000 005 1 000e 008 L L 123 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 Cycle Number Selected Detector All Well s 1 12 Documen t MycoP Linear Intra 240811 sds Standard Curve Figure 2 Post run data analysis amplification graph in logarithmic scale RQ S47 48 EN doc Qanaumca 2 0 lica it www abanaliti Standard Curve 33 227 32 000 30 000 Ct 28 000 26 000 24 000 23 161 2 000 3 000 4 000 5 000 Log Detector Myco P Slope 3 302319 Intercept 39 756
5. P Purple 1 27 uL 2 27 ul 4 x 27 uL the B globin gene BG STORE AT 2 C 8 C TUBE T DESCRIPTION LABEL OR LID COLOR DNA containing a part of the POSITIVE Mycoplasma pneumoniae CONTROL Purple 1 30 ul 1 60 ul 1 110 ul genome positive control MYCO P DNA containing a part of the POSITIVE B globin gene CONTROL Blue 1 30 ul 1 60 ul 1 110 ul positive control BG DNA containing a part of the B globin gene ve 2x125ul 4x125 ul 8x 125 ul CONTROL internal control RQ S47 48 EN doc 4 a www abanalitica it 3 STORAGE AND STABILITY OF REAGENTS Each component of the kit must be stored according to the directions indicated on the label of each box In particular Box F Store at 30 C 20 C Box F Store at 2 C 8 C If stored at the recommended temperature all test reagents are stable until their expiration date In order to avoid degradation of the reagents the 2X EV Real Time Mix and the Oligomix should not undergo more than two freeze thaw cycles The 2X EV Real Time Mix contains fluorescent molecules therefore it is recommended to store it protected from direct light If performing runs with low numbers of samples it is recommended to aliquot the reagents beforehand Qanaumca 5 RQ S47 48 EN doc www abanalitica 4 PRECAUTIONS FOR USE The kit must be used only as an IVD and handled by qualified technicians
6. Sodium Hypochlorite 547 48 EN doc 6 Qanauitica www abanalitica it 5 SAFETY RULES 5 1 GENERAL SAFETY RULES e Wear disposable gloves when handling reagents and clinical samples Wash hands after the procedure e Do not pipette by mouth e No known diagnostic method can assure the absence of infective agents Therefore consider every clinical sample to be potentially infectious and handle it accordingly e All devices that come into contact with clinical samples must be considered contaminated and disposed of as such In case of accidental spilling of the samples clean up with 10 Sodium Hypochloride The materials you use to clean must be disposed of in special containers for contaminated products e Clinical samples materials and contaminated products must be decontaminated before disposal We recommend to use one of the following decontamination methods immerse for 30 minutes in a solution of 5 Sodium Hypochlorite 1 volume of 5 Sodium Hypochlorite solution on 10 volumes of contaminated fluid OR autoclave at 121 C for at least 2 hours ATTENTION Do not autoclave solutions containing Sodium Hypochlorite 5 2 SAFETY RULES CONCERNING THE KIT The risks for the use of this kit are related to the single components Dangerous components none The Material Safety Data Sheet MSDS of this device is available upon request 7 RQ S47 48 EN doc www abanalitica 6 MATERIAL REQUIRED
7. kit allows to detect the presence of reaction inhibitors and to monitor the extraction process by amplification of the B globin gene amplification control in multiplex with the target pathogen This is a valuable tool for identifying false negative results In cellular samples the endogenous gene is amplified For acellular specimens an internal control is added which consists of recombinant DNA containing the B globin gene The kit includes a ready to use Mastermix and an Oligomix The Oligomix contains the specific primers and probes whereas the Mastermix contains all reagents necessary for the amplification reaction as well as the following components e ROX is an inert colorant that exhibits stable fluorescent properties throughout all amplification cycles On some Real time PCR instruments it is used for normalization in order to compensate for differences between wells caused by pipetting errors or instrument limitations e The dUTP UNG system prevents contamination from previous amplification runs The dUTPs are used to incorporate uracil residues into amplicons during amplification sessions Before each new run the UNG enzyme degrades any single or double stranded DNA containing uracil This way any amplification products from former sessions are eliminated 547 48 EN doc Qanauitica 1 2 www abanalitica it 10 SAMPLE COLLECTION MANIPULATION AND PRETREATMENT Commonly Mycoplasma pneumoniae is identified from samples from o
8. 355 R2 0 999270 Document MycoP Linear Intra 240811 sds Standard Curve Figure 3 Post run data analysis standard curve ANALITICA 24 RQ S47 48 EN doc 12 TROUBLESHOOTING Absence of amplification signals for positive controls standards and samples The instrument was not programmed correctly Repeat the amplification taking care of the instrument programming Pay particular attention to the thermal profile the fluorophores selected and that the position of the samples in the plate protocol corresponds to the positions on the actual plate The amplification mix was not prepared correctly Prepare a new amplification mix making sure to follow the instructions given in paragraph 11 3 The kit was not stored properly or it was used past the expiration date Check both the storage conditions and the expiration date reported on the label Use a new kit if needed Weak amplification signal for positive controls standards Positive controls standard solutions were stored incorrectly and have degraded Store the positive controls standard solutions correctly at 2 8 C and make sure that they do not undergo any freeze thaw cycle as well Do not use the positive controls standards past the expiration date The reaction mix does not function correctly Make sure to store the 2X EV Real Time Mix and Oligomix correctly at 20 C 30 C Avoid unnecessary freeze thaw cycles 547 48 EN doc Qanauitica 2 2 www abanali
9. ANALITICA ADVANCED BIOMEDICINE User manual REALQUALITY RS MYCO P code RQ S47 Kit for the identification and quantification of Mycoplasma pneumoniae IVD RQ S47_48_EN doc 10 11 PRODUCT INFORMATION 1 1 Intended use KIT CONTENT STORAGE AND STABILITY OF REAGENTS PRECAUTIONS FOR USE SAFETY RULES 5 1 General safety rules 5 2 Safety rules concerning the kit MATERIAL REQUIRED BUT NOT PROVIDED 6 1 Reagents 6 2 Instruments 6 3 Disposables INTRODUCTION TEST PRINCIPLE PRODUCT DESCRIPTION SAMPLE COLLECTION MANIPULATION AND PRETREATMENT PROTOCOL 11 14 DNA Extraction 11 2 Internal control 11 3 Instrument programming 11 4 PROTOCOL FOR QUALITATIVE ANALYSIS 11 5 PROTOCOL FOR QUANTITATIVE ANALYSIS 11 6 Qualitative analysis and interpretation of results 11 7 Quantitative analysis and interpretation of results anauitica 1 aee A o it www abanalitica 10 12 13 14 14 14 15 16 17 18 20 RQ S47 48 EN doc 12 TROUBLESHOOTING 13 DEVICE LIMITATIONS 14 DEVICE PERFORMANCES 14 1 14 2 14 3 14 4 14 5 14 6 14 7 Analytical specificity Analytical sensitivity detection limit Analytical sensitivity linearity Reproducibility Diagnostic specificity Diagnostic sensitivity Accuracy 15 REFERENCES 16 RELATED PRODUCTS RQ S47 48 EN doc 2 22 24 24 24 24 25 25 26 26 27 27 28 anauitica eterna ica it www ab
10. UCT DESCRIPTION on page 12 for all positions wells in use Enter where required 25 ul as final reaction volume ANALITICA es 1 5 RQ S47 48 EN doc www abanaliti 11 4 PROTOCOL FOR QUALITATIVE ANALYSIS Once thawed mix the reagents by inverting the tubes several times do not vortex then centrifuge briefly Prepare the reaction mix rapidly at room temperature or work on ice or ona cooling block Try when possible to work in an area away from direct light Prepare as described below a mix sufficient for all the samples to be tested counting also the positive and negative control in the latter H2O is added instead of DNA and when calculating the volume consider an excess of at least one reaction volume 2X EV Real Time mix 12 5 ul Oligo Mix MYCO P 1 uL Total volume 20 uL Mix by inverting the tubes several times in which the mix was prepared in and then centrifuge briefly Pipette 20 pL of the mix in each well on the plate Add 5 uL of extracted DNA to each well or 5 uL of positive control DNA in the correct position on the plate Always amplify a negative control together with the samples to be analyzed add sterile water instead of extracted DNA to the corresponding well Hermetically seal the plate by using an optical adhesive film or the appropriate sealer Make sure that there are no air bubbles in the bottom of the wells and or centrifuge the plate at 4000 rpm for about 1 minute L
11. analiti 1 PRODUCT INFORMATION 1 1 INTENDED USE The REALQUALITY RS MYCO P is an IVD device for the identification of Mycoplasma pneumoniae MYCO P by amplification of the gene coding cytoadhesion P1 If used together with the REALQUALITY RS MYCO P STANDARD code RQ 48 ST it allows the quantification of the number of bacterial genome copies present in the sample This kit uses Real Time PCR amplification starting from DNA extracted from human clinical samples This in vitro diagnostic test allows the detection and quantification of M pneumoniae constituting an auxiliary device for diagnosis and monitoring of M pneumoniae infections It is recommended to use this kit as indicated in the instructions herein This manual refers to the following product REALQUALITY RS MYCO P Kit for the identification and quantification of Mycoplasma pneumoniae This product is in accordance with 98 79 CE Directive Annex III regarding the in vitro medical diagnostic devices CE mark Contains all the reagents needed for Real Time amplification Code Product PKG RQ S47 48 REALQUALITY RS MYCO P 48 test RQ S47 96 REALQUALITY RS MYCO P 96 test 3 RQ S47 48 EN doc www abanalitica 2 KIT CONTENT STORE AT 30 20 TUBE T DESCRIPTION LABEL OR LID COLOR 2X EV ZA Mastermix Real Time Mix 1 340uL 2x340 uL 4 340 uL Primer and probe Mix for amplification of MYCO P and Oligomix MYCO
12. ary atypical pneumonia which mainly occurs in children and in youths before 30 years of age M pneumoniae is an obligate aerobe bacterium which adheres to epithelium ciliatum by particular adhesins causing ciliatosis block of cilium and afterwards can cause epithelium destructions and therefore irritation and coughing The infection in the majority of cases is asymptomatic however there have been reported cases of severe types pneumonia with haematological and neurological involvement in immunocompromised patients Respiratory tract infections caused by this bacterium are however a major cause of other pathologies in addition to pneumonia as tracheobronchitis pharyngitis and asthma When this bacterium moves to other parts of the body it can be associated to various manifestation non pulmonary pathology involving central nervous system liver pancreas blood skin and joints M pneumoniae is transmitted by coughing saliva droplets People with an active infection of M pneumoniae can carry the bacterium in the nose throat and sputum indicating a general involvement The traditional diagnosis of M pneumoniae infections is difficult since serological and cultural methods require long times making it impossible to prescribe an effective treatment It is difficult to be culture Micoplasma in laboratory and frequently for this reason it cannot be identified as pathological cause of the diseases Today the most commonly reliab
13. er s fluorescence is blocked Upon binding to a target sequence the quencher and reporter become separated and the light emitted by the reporter can be detected Typically the main part of a Real time PCR run consists of several 30 50 amplification cycles The higher the initial concentration of an amplified sequence the earlier the PCR produces an amplicon concentration that displays a fluorescence clearly distinguishable from the background Thus the initial concentration of a target sequence can be determined Specific for each reaction is the so called Ct value or threshold cycle It is defined as the point or cycle at which the fluorescence signal becomes clearly distinguishable from the background while the PCR is still in the exponential amplification phase The latter condition makes sure the number of amplicons is proportional to the number of reaction cycles passed Using a standard curve the initial concentration of a target sequence can be calculated The standard curve is established by amplifying standard samples with known concentrations of the target sequence A thermocycler equipped with a corresponding detector can record the fluorescence events and thus monitor the reaction in real time 547 48 EN doc Qanauitica 1 0 www abanalitica it The initial concentration of the target in the samples is determined by comparing the Ct value of each sample with a standard curve that was created by amplifying standards with k
14. he expected results RESULT INTERPRETATION B globin positive control Amplification signal present No amplification signal Correct B globin amplification Amplification problems repeat the analysis B globin negative control No amplification signal No contamination Amplification signal Contamination repeat the analysis RESULT INTERPRETATION MYCO P positive control Amplification signal present Correct MYCO P amplification No amplification signal Amplification problems repeat the analysis MYCO P negative control No amplification signal No contamination Amplification signal Contamination repeat the analysis RQ S47_48_EN doc 18 anauitica steeped Lea www abanalitica it B globin detector MYCO P detector INTERPRETATION Amplification signal Amplification signal No amplification signal Sample positive for MYCO P sample negative for MYCO P No amplification signal Amplification signal sample positive for MYCO P No amplification signal Sample not suitable Repeat the DNA extraction ATTENTION The assay was standardized in order to favour the target pathogen amplification reaction Therefore the amplification signal of the B globin gene fluorescence in JOE can have a delayed or absent Ct in MYCO P positive samples anauitica aee d ou t www abanalitica
15. ificity of this device was calculated to be 100 14 6 DIAGNOSTIC SENSITIVITY A significant number of samples positive for Mycoplasma pneumoniae were tested simultaneously with the REALQUALITY RS MYCO P kit and another CE IVD device or reference method From the obtained results the diagnostic specificity of this device was calculated to be 100 547 48 EN doc Qanauitica 2 6 www abanalitica it 14 7 ACCURACY The accuracy was calculated as the number of correct amplifications over the total number of executed amplifications The REALQUALITY RS MYCO P kit has an accuracy of 100 15 REFERENCES Balin BJ et al Med Microbiol Immunol Berlin 187 23 42 1998 Dowell SF et al Clin Infect Dis 33 492 503 2001 Gieffers JE et al J clin Microbiol 38 881 882 2000 Grayston JT Clin Infect Dis 155 757 761 1992 Gupta S et al Circulation 96 404 407 1997 Hammerschlag MR Infect Dis Clin Practice 8 232 240 1999 Hammerschlag MR Curr Infect Dis Rep 2 115 120 2000 Hammerschlag MR et al J Clin Microbiol 38 4274 4276 2000 Kuo CC et al Clin Microbiol Rev 8 451 461 Kuo CC et al Arterioscler Thromb 10 1501 1504 1993 Maurin M et al J clin Microbiol 35 2283 2287 1997 Muhlestein JB et al Circulation 97 633 636 1998 Normann E et al Pediatr Infect Dis J 18 72 73 1999 Peeling RW et al J Infect Dis 181 Suppl 3 5426 5429 2000 Ramirez JA Ann Intern Med 125 979 982 1996 Saiki RK et al Science 230 1350
16. le diagnostic method is he polymerase chain reaction PCR With Real time PCR it allows for a rapid and reliable quali quantitative diagnosis 9 RQ S47 48 EN doc www abanalitica 8 TEST PRINCIPLE The PCR method Polymerase Chain Reaction was the first method of DNA amplification method described in literature Saiki RK et al 1985 It is can be defined as an in vitro amplification reaction of a specific part of DNA target sequence by a thermostable DNA polymerase This technique was shown to be a valuable and versatile instrument of molecular biology its application contributed to a more efficient study of new genes and their expression and has revolutionized the fields of laboratory diagnostics and forensic medicine The Real time PCR represents an advancement of a basic research technology providing the possibility to determine the number of amplified DNA molecules amplicons during the polymerase chain reactions The monitoring of amplicons is based on primers or probes labeled with fluorescent molecules molecular beacon scorpion primer etc These primers or probes usually contain a fluorophore reporter and a molecule that blocks the reporter s specific fluorescence quencher Fluorescent emission is determined by the relative proximity of the reporter molecule to the quencher While a primer or probe are not bound to a target sequence their reporter and quencher are in close proximity and the report
17. nown concentrations Figure 1 Correlation Coefficient 0 999 Slope PCR Efficiency 99 3 Threshold Cycle ENRON 06 DOSY RO OO OT Aig NN V 02 46 8 10 12 14 16 18 20 22 D 28 30 32 34 36 38 40 42 44 46 el 438 Intercept 8 768 Y 3 338 X 38 N68 n Unknowns Standards Log Starting Quantity copy number Figure 1 Creating a standard curve using standards with known concentrations The main advantage of Real time PCR compared to conventional techniques of amplification is the possibility to perform a semi automated amplification This means the extra steps necessary to visualize the amplification result can be avoided and the risk of contamination by post PCR manipulation is reduced anauitica ADVANCED BIOMEDICINE www abanalitica it 11 RQ S47 48 EN doc 9 PRODUCT DESCRIPTION The REALQUALITY RS MYCO P kit code RQ S45 is an IVD device for the identification of Mycoplasma pneumoniae by amplification of the gene coding cytoadhesion P1 If used together with REALQUALITY RQ MYCO P STANDARD code RQ 48 ST it allows the quantification of the number of bacterial genome copies present in the sample The respective standard curve consists of 4 points from 10 to 10 genome copies per reaction The positive controls supplied in this kit contain DNA fragments that correspond to the genetic region of interest and as such these controls are not dangerous for the user The
18. oad the plate on the instrument making sure to position it correctly and start the amplification cycle RQ S47 48 EN doc 16 Qanaumca www abanaliti 11 5 PROTOCOL FOR QUANTITATIVE ANALYSIS The quantitative analysis can be performed by using REALQUALITY RQ MYCO P STANDARD code RQ 48 ST Follow the instructions reported in the previous paragraph to prepare a reaction mix sufficient for the standard curve A negative amplification control must be included on the plate in which is added instead of DNA Aliquot 20 pL of the mix in each well on the plate Add 5 uL of extracted DNA to each well or 5 uL of each quantification standard dilution in the corresponding positions on the plate Hermetically seal the plate by using an optical adhesive film or the appropriate sealer Make sure that there are no air bubbles in the bottom of the wells and or centrifuge the plate at 4000 rpm for about 1 minute Load the plate on the instrument making sure to position it correctly and start the amplification cycle 17 RQ S47 48 EN doc www abanalitica 11 6 QUALITATIVE ANALYSIS AND INTERPRETATION OF RESULTS At the end of the reaction view the graph in logarithmic scale Analyze the MYCO P and f globin amplification results separately by selecting the correct detector and use the following instructions for interpretation Before considering the sample results make sure that the positive and negative controls have t
19. of your extraction system The sample is suitable for analysis if the resulting Ct value of the internal control is lt 35 This threshold is only valid if the internal control was as added as described above ratio of 1 6 Applied Biosystems 7500 Fast Dx Real time PCR System Threshold 0 05 For further information please contact AB ANALITICA s technical support 547 48 EN doc Qanauitica 1 4 www abanalitica it 11 3 INSTRUMENT PROGRAMMING 11 3 1 Create a thermal profile Use the following thermal profile Cycle Repeats Step Time UNG Activation 1 1 1 02 00 50 0 Taq Activation 2 1 1 10 00 95 0 Amplification 3 45 1 00 15 95 0 cycles 60 Fluorescence detection step 11 3 2 Plate Setup On the grid of the new plate set up the positions for the negative control NTC positive control or standards STD and samples Unknown Quantitative Analysis For Quantitative Analysis enter the corresponding concentrations of the MYCO P standards 10 10 10 and 10 bacteria gene copies reaction Note Make sure the position is the same as on the plate and mark each sample with its name Select the following the detectors for MYCO P and BG as follows Name Reporter dye Quencher dye MYCO P FAM none p globin JOE none ATTENTION Some instruments require to also select the fluorophore ROX see 9 PROD
20. order to determine the cross reactivity of this device samples positive for other potentially cross reactive pathogens or pathogens from the anatomical region as Mycoplasma pneumoniae was identified in were amplified with this device None of the tested pathogens gave a positive signal with the IVD device 14 2 ANALYTICAL SENSITIVITY DETECTION LIMIT Serial dilutions of quantification standard ranging from 1 to 0 05 copies of viral genome copies uL were tested in three consecutive experiments in order to determine the analytical sensitivity For each dilution 5 uL were amplified in eight replicates per run in multiplex with the internal control The results were analyzed by Probit analysis as illustrated in the graph reported in Figure 4 The limit of the analytical sensitivity for the REALQUALITY RS MYCO P p 0 05 kit is reported in Table 1 547 48 EN doc Qanauitica 2 4 www abanalitica it 1 0 5 95 Probabilita 0 4 0 6 0 8 0 2 0 0 ProbitiDose Figure 4 Graph of the Probit analysis results for determination of analytical sensitivity for the REALQUALITY RS MYCO P kit on Applied Biosystems 7500 Fast DX Real Time PCR System expressed in bacteria genome copies reaction 14 3 ANALYTICAL SENSITIVITY LINEARITY The linearity of the assay was determined using a quantification standard panel The results of the analysis are reported in Table 1 with the linear regression 14 4 REPRODUCIBILITY Intra assay
21. ro and nasopharyngeal swabs spit bronchoalveolar lavage BAL bronchial aspirate sputum but also pulmonary biopsies and PBMC peripheral blood mononuclear cells The device was tested on DNA extracted from nasopharyngeal swabs spit saliva sputum PBMC and FFPE tissue Respiratory samples can be liquefied by adding 1 5 N acetyl L cysteine NAC or 0 1 dithiothreitol DTT Use sterile disposable sample collectors with a screw cap Samples must be stored at 2 C 8 C not longer than 48 hours or at 30 C 20 C for a longer period some months FFPE samples as well as fresh or frozen histological samples must be disrupted mechanically e g using a sterile scalpel and then treated with lysis buffer and proteinase K FFPE histological samples formalin fixed paraffin embedded have to be deparaffinized before digestion PBMC have to be separated from whole peripheral blood by density gradient centrifugation e g Ficoll Hypaque Sample collection should follow the common sterility precautions The samples have to be transported in sterile boxes without transport medium Blood must be treated with EDTA Other anticoagulation agents as heparin are strong inhibitors of TAQ polymerase and can impair the Real Time PCR If processed within 4h after the collection fresh blood can be stored at 2 C 8 C Otherwise it must be frozen at 30 C 20 C ANALITICA es 1 3 RQ S47_48_EN doc www abanalitica 11 PROTOCOL 11
22. tica it Amplification signal of B globin very delayed or absent in the extracted sample MYCO P negative e The extracted DNA was not suitable for amplification and the amplification reaction was inhibited Make sure to extract the nucleic acids correctly f an extraction method uses wash steps with solutions containing Ethanol make sure no Ethanol residue remains in the DNA sample Use the extraction methods suggested in paragraph 11 1 For any further problems please contact AB ANALITICA s technical support at laboratorio abanalitica it fax 39 049 8709510 or tel 39 049 761698 ANALITICA E Qu 23 RQ S47 48 EN doc www abanalitica 13 DEVICE LIMITATIONS The kit can have reduced performance if e The clinical sample is not suitable for this analysis sampling and or storage error i e blood treated with anticoagulants other than EDTA like heparin etc e DNA is not suitable for amplification due to the presence of amplification reaction inhibitors or to the use of inappropriate extraction method e The kit was not stored correctly 14 DEVICE PERFORMANCES 14 1 ANALYTICAL SPECIFICITY The specificity of the REALQUALITY RS MYCO P code RQ S45 kit is guaranteed by an accurate and specific selection of primers and probe and also by the use of stringent amplification conditions Alignment of primers and probes in the most important databanks showed no non specific pairing In
23. variability In order to determine the variability among replicates of the same sample in the same assay a dilution of 50 transcript copies uL of the quantification standard corresponding to a final amount of 250 copies reaction was amplified in eight replicates in one run The intra assay variability coefficient of the method in respect to the Cycle threshold Ct is reported in the table below ANALITICA auus 2 5 RQ S47 48 EN doc Inter assay variability In order to determine the variability of replicates of the same sample in different runs the least concentrated quantification standard 20 transcript copies uL corresponding to a final amount of 100 copies reaction was amplified in duplicates in three consecutive runs For each run the variability coefficient was calculated from the Ct of the samples The inter assay variability coefficient was calculated from the average of the variable coefficients of each run and is reported in the table below ABI 7500 Fast Dx ABI 7300 StepOnePlus Detection limit bacterial genome copies uL probit p 0 05 Range of linearity bacterial genome copies reaction Intra assay variability 96 Inter assay variability 96 14 5 DIAGNOSTIC SPECIFICITY A significant number of samples negative for MYCO P were tested simultaneously with the REALQUALITY RS MYCO P kit and another CE IVD device or reference method From the obtained results the diagnostic spec
24. who are trained in molecular quantitative biology techniques applied to diagnostics Before starting the kit procedure read the user manual carefully and completely Keep the kit away from heat sources Please pay particular attention to the expiration date on the label of each box do not use any part of the kit past the expiration date The reagents present in the kit must be considered an undividable unit Do not use them separately or in combination with reagents from other kits or lots All frozen reagents must be thawed at room temperature before use Do not vortex Mix the solutions by inverting the tubes several times then centrifuge briefly Work swiftly particularly if preparing the reactions at room temperature If possible work on ice or on a cooling block In case of any doubt about storage conditions box integrity or application of the method please contact AB ANALITICA s technical support at laboratorio abanalitica it During nucleic acid amplification the technician has to take the following special precautions Use filter tips In order to avoid contamination store the biological samples the extracted DNA amplified DNA and the positive controls included in the kit separated from the Mastermix and the Oligomix es Set up pre and post PCR work areas do not share instruments or consumables pipettes tips tubes etc between those areas Change gloves frequently Wash the bench surfaces with 5
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