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Nucleic acid Amplification Assay Development using the BD MAX Sue C. Kehl, Ph.D. D(ABMM) Associate Professor, Pathology Medical College of Wisconsin Associate.

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Presentation on theme: "Nucleic acid Amplification Assay Development using the BD MAX Sue C. Kehl, Ph.D. D(ABMM) Associate Professor, Pathology Medical College of Wisconsin Associate."— Presentation transcript:

1 Nucleic acid Amplification Assay Development using the BD MAX Sue C. Kehl, Ph.D. D(ABMM) Associate Professor, Pathology Medical College of Wisconsin Associate Director, Clinical Pathology Technical Director, Microbiology and Molecular Infectious Disease Childrens Hospital of Wisconsin

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3 Assay Development Primer selection Amplification and Detection – Coverage Assay optimization – Multiplex – Protocol – Threshold Extraction Validation – Dynamic Range – Efficiency – Analytical Sensitivity and Specificity – Clinical Sensitivity and Specificity – Precision Quality Assurance

4 Identification of Primers Commercially available Published – Chlamydia pneumoniae Outer membrane protein gene – Mycoplasma pneumoniae 16s rRNA gene – Legionella pneumophila and Legionella micdadei mip gene Developed internally in collaboration with Midwest Respiratory Virus Program – RSV polymerase (L) gene – Influenza A matrix (M) gene – Influenza B matrix (B) gene – Human Metapneumovirus polymerase (L) gene target – Parainfluenza

5 Serial dilutions of RSV stock detected with expected melts

6 Serial dilutions of Influenza A

7 Serial dilutions of Influenza B stock detected with expected melts

8 Coverage Document coverage of circulating strains Essential step for commercially available, published or internally developed Examined circulating RSV types from collection of clinical isolates in addition to ATCC strains

9 Optimize primer/probe concentrations

10 Optimize Assay Components Optimize enzyme system Started with Quantitect and switched to Invitrogen – Platinum Tfi - a recombinant Tfi DNA polymerase complexed with a proprietary antibody mix that inhibits polymerase activity at ambient temperature, allowing room-temperature reaction setup. Activity is restored after the initial denaturation step in PCR cycling at 94 C, providing an automatic hot start for increased specificity, sensitivity, and yield. – Superscript III reverse transcriptase May want to consider a one step (rTth DNA polymerase)

11 Protocol optimization TIME (s)TEMP UNG RT DENATURE PCR X 45 DENATURE 1595 ANNEAL 3056 EXTENSION 3076 DENATURE 1595 MELT Previous protocol Predicted time: 1.7 hrs Actual time: > 2.5 hrs Altered protocol to decrease time Eliminate UNG Decrease RT time Shorten melt range Actual time: 2 hours

12 Multiplex Assay Prepare Multiplex Repeat Limit of Detection RSV

13 Influenza A Influenza B

14 Cross Reactivitiy High titer virus – no bleed through in other channels High titer of multiple viruses – accurate detection of all viruses No assay interference or loss in sensitivity from internal control

15 RSV Cross Talk

16 RSV Cross Talk Elimination

17 Sample How much sample to add to lysis buffer? Dependent upon sample type to be tested and desired analytical sensitivity – Sensitive assay without sample interference Respiratory samples in M4 transport medium – Nasopharyngeal swabs – Bronchial alveolar lavage Serially diluted stock strains into known negative clinical samples

18 Extraction Extractions were performed in triplicate by the following methods. Qiagen BioRobot EZ1 - EZ1 Virus Mini Kit (Qiagen Inc., Valencia, Ca) was used as recommended by the manufacturer. To improve the analytical sensitivity cRNA (8 µg/reaction mix) was added to the extraction cartridges. 400 µl of RSV and Influenza were extracted into 60 µl. Nuclisens easyMAG µl of RSV and Influenza was added to 1 ml lysis buffer and extracted using the NucliSens easyMAG (bioMerieux, Dunham, NC ) as recommended by the manufacture with final elution volume of 55 µl BD MAX–Influenza A and RSV were extracted using BD Max RNA-3 extraction reagent strips. 475 µl of sample was added to 700 µl of the Specimen Processing tubes. Approximately 900µl is then eluted into 10 µl.

19 Ct value ManualeasyMAGEZ1Singleplex BD MAX RSV TCID 50 /ml INFLUENZA A 10 1 TCID 50 /ml INFLUENZA B 10 1 TCID 50 /ml

20 Contamination High concentration of virus – Tested alternating locations in two runs No evidence of contamination or carryover

21 Threshold Most of the assay development had used instrument calculated threshold (-1.6) Specimens were considered positive if Ct < 40 with an appropriate Tm

22 Clinical testing showed samples with Ct < 40 and no Tm.

23 Threshold Based on calculations, a cutoff of -1, should have allowed us to detect all positives with appropriate melts at a Ct < 40 In reality, a significant loss in sensitivity was observed

24 Moving the threshold to -1.2, still resulted in a minor loss of analytical sensitivity, but allowed for better discrimination between true positives and negatives

25 Assay validation – Document dynamic range Desire at least 6 log range Test diluted stock in triplicate – Analytical sensitivity Serial dilutions of quantitated stock in viral transport medium and negative clinical sample to determine limit of detection and dynamic range – Analytical specificity Similar organisms Other organisms expected in sample type – Clinical sensitivity and specificity 20 positives clinical samples for each virus 40 negatives clinical samples for each virus Calculate accuracy of assay – Interpretation Ct value for positive Tm for identification – Precision – Lack of inhibition

26 Table 1. Comparison of frozen tissue culture specimens in Multiplex RT-PCR assays on Two color BD MAX ProFluV12.2 N=92*93 Indeterminants or Unresolved67 Sensitivity (95% CI) Specificity (95% CI) Sensitivity (95% CI) Specificity (95% CI) FluA N=16 93% (66-100) 100% (95-100) 100% (74-100) 100% (96-100) FluB N=21 80% (56-94) 100% (95-100) 90%*** (70-99) 100% (95-100) RSV N=20 95% (73-100) 95% (87-98) 100% (83-100) 96% (100) Total 88% (77-96) 99.1% (97-100) 96% (87-100) 99.1% (97-99) *two samples had insufficient material for testing ***1/2 samples not detected were RSV/FluB dual positives and correctly identified as RSV.

27 Table 2. Virus recovery from fresh specimen in tissue culture V12.2Tissue Culture N=57 Indeterminants/Unresolved0NA Virus Recovered No. (%) FluA2 (3.5%)0 (0%) FluB7 (12%)8 (14%) RSV11 (19%)1 (2%) Negative37 (65%)46 (81%)* * Two specimens were positive by DFA for Para/Adeno

28 Precision Ct Test known concentration 5 times within a run Test known concentration over 10 days Reasonable expectation is log, which correlates to 5% Our assay showed coefficient of variation of 2.1 – 2.7%

29 Precision Tm HMPV AHMPV BSOIVRSVFLU B Minimum % Percentile Median % Percentile Maximum Mean Std. Deviation Std. Error Lower 95% CI of mean Upper 95% CI of mean Coefficient of variation1.25%1.52%1.31%1.39%2.21%

30 Quality Assurance Test first 5 – 10 clinical tissue culture isolates of each season in the PCR assay to assure detection Monitor in silico coverage of primers and probes

31 Subtyping – Pandemic 2009 H1N1 had been accurately detected by our previous assay – Based on published sequences, developed primers and probes to 2009 H1N1, H3N2 Primers and probes to hemagglutinin gene H3 labeled with FAM 475 H1N labeled with Cal Red 585 – Document dynamic range – Determine analytical sensitivity – Determine analytical specificity – Determine clinical sensitivity and specificity

32 Limit of detection – H1N – 10 1 TCID 50 /ml with Tm – H3N TCID 50 /ml with Tm

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35 Additional Assays Mycoplasma, Chlamydia Multiplex Assay – Utilizes published primers and probes – Chlamydia pneumoniae Outer membrane protein gene Chlamydia probe – FAM 475 – Mycoplasma pneumoniae 16s rRNA gene probe- MAXN 530 Legionella Singleplex Assay – Legionella pneumophila and Legionella micdadei mip gene Probe - FAM 475 – BD DNA Extraction Kit DNA-3 – BD Master mix with Cal Red Internal Control

36 Mycoplasma pneumoniae Chlamydophila pneumoniae LOD = 10 0 pfu/ml LOD = 100 copies/ml

37 Multiplex assay LOD = pfu/ml LOD = 28 copies/ml

38 Optimize Specimen

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40 Legionella

41 Optimize primer probe concentration

42 Throughput

43 Nested PCR Runs

44 Reliability Two instruments – 73 – Service once in 6 months due to lane loading issues – 90 – Service once in 6 months related to software upgrade Software upgrade in April and August Performance related to lane loading issues or failed extractions Lot 1 CM0012 – 2.3% Lot 2 CM0012 – 4.6% Lot 2 CM0073 – 2.2% Lot 3 CM0073 – 1.8% Lot 4 CM % Lot 4 CM %

45 Quality Control New Quantitated virus stocks – Test 5 times in assay – Determine acceptable Ct range (mean Ct S.D.) – Monitor 20 assays – Recalculate acceptable Ct range Daily – Rotate positive control among all viruses – Negative (No template) control Monthly – Monitor actual mean and standard deviation and assess for difference over time

46 Statistical analysis of QC H1N1 SOIV One-way analysis of variance P value P value summaryns Are means signif. different? (P < 0.05)No Number of groups9 F1.79 R squared ANOVA TableSSdfMS Treatment (between columns) Residual (within columns) Total No post tests. P > 0.05

47 New primers, probes, enzyme – Prior to use – Test all controls – Document within acceptable range New extraction kits – Prior to use – Test all controls – Document within acceptable range

48 Assay Development Primer selection Amplification and Detection – Coverage Extraction Assay optimization – Multiplex – Protocol – Threshold Validation – Dynamic Range – Efficiency – Analytical Sensitivity and Specificity – Clinical Sensitivity and Specificity – Precision Quality Assurance and Quality Control

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50 References Khanna, M., J. Fan, K. Pehler-Harrington, C. Waters, P. Douglass, J. Stallock, S. Kehl, K.J. Henrickson The pneumoplex assays, a multiplex PCR-enzyme hybridization assay that allows simultaneous detection of five organisms, Mycoplasma pneumoniae, Chlamydia (Chlamydophila) pneumoniae, Legionella pneumophila, Legionella micdadei, and Bordetella pertussis, and its real-time counterpart. J. Clin Microbiol Feb;43(2): Bose, M.E., E.T. Beck, N. Ledeboer, S.C. Kehl, L.A. Jurgens, T. Patitucci, L. Witt, E. LaGue, P. Darga, J. Hie, J. Fan, S. Kumar, K.J. Henrickson Rapid semi- automated subtyping of influenza during the 2009 swine-origin Influenza A H1N1 epidemic in Milwaukee, Wisconson. J Clin Microbiol. 47(9): Kehl, S.C., L. Jurgens, M. Bose, E. Beck. J. Fan, S. Kumar, L. Witt, T. Patitucci, P. Darga, E. LaCue, K. Wilkinson, K.J. Henrickson Comparison of a novel multiplex automated laboratory-developed RT-PCR assay for RSV and Influenza A/B with the ProFLU+ Assay. M th Annual Clinical Virology Symposium. Henrickson, K.J., L. Jurgens, M. Bose, E.T. Beck, S.C. Kehl, J. Fan, S. Kumar, L. Witt, T. Patitucci, P. Darga, E. LaGue Rapid automated detection of Parainfluenza virus types 1, 3 and human Metapneumovirus (hMPV) by multiplex real time RT-PCR using two new methods and novel primer/probe chemistry. M th Annual Clinical Virology Symposium. Beck, E.T., L. Jurgens, M. Bose, S. Kehl, J. Fan, S. Kumar, L. Witt, T. Patitucci, P. Darga, L. LaGue, K.J. Henrickson Rapid automated detection of Influenza A, Influenza B, and RSV A/B by multiplex real time RT-PCR using two new methods and novel primer/probe chemistry. M th Annual Clinical Virology Symposium.

51 References Kehl, S.C., S. Goodacre, K. Vaughn, N. Ledeboer, K.J. Henrickson Comparison of the Jaguar System, the Qiagen Biorobot EZ1 and the NucliSens easyMAG for extraction of Enterovirus, RSV and Influenza A. M th Annual Clinical Virology Symposium. Beck, E.T., L.A. Jurgens, S.C. Kehl, M.E. Bose, T. Patitucci, E. LaGue, P. Darga, K. Wilkinson, L.M. Witt, J. Fan, J. He, S. Kumar, K.J. Henrickson Rapid automated detection of Influenza A, Influenza B, and RSV A/B by multiplex real-time RT-PCR during the 2009 H1N1 swine-origin influenza virus (S-OIV) epidemic in Milwaukee, Wisconsin. J. Mol. Diagn : 74-81; doi: /jmoldx Trost, J.F., E. T. Beck, M. E. Bose, K. Waant, J.Fan, S. Kumar, S. C. Kehl, K. J. Henrickson Fully-automated multiplex Real-Time RT-PCR for identification and Typing of Parainfluenza S th Annual Clinical Virology Symposium Trost, J.F., E. T. Beck, M. E. Bose, M. Nowak, J.Fan, S. Kumar, S. C. Kehl, K. J. Henrickson Fully-automated multiplex Real-Time RT-PCR for identification Of Influenza A, Influenza B, RSV, and hMPV. M th Annual Clinical Virology Symposium


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