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
Children’s Hospital of Wisconsin

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
300 40 RT
1800 50
DENATURE
120 95
PCR X 45
‘ DENATURE 15
‘ ANNEAL 30 56
‘ EXTENSION 76
MELT
40-95
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 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 µl of sample was added to 700 µl of the Specimen Processing tubes. Approximately 900µl is then eluted into 10 µl.

19. Ct value
Manual
easyMAG
EZ1
Singleplex
BD MAX
RSV TCID 50/ml
37.6
36.8
35.0 37
36.3
INFLUENZA A 10 1 TCID 50/ml
32.4
33.7
31.2 36
INFLUENZA B 10 1 TCID 50/ml
33.0
31.8
34.6
35.3
34.9

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
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 Analytical sensitivity
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
ProFlu
V12.2 N=
92* 93
Indeterminants or
Unresolved 6 7
Sensitivity (95% CI)
Specificity (95% CI)
FluA N=16
93%
(66-100)
100% (95-100)
100%
(74-100)
(96-100)
FluB N=21
80%
(56-94)
(95-100)
90%*** (70-99)
RSV N=20
95%
(73-100)
(87-98)
(83-100)
96%
(100)
Total
88%
(77-96)
99.1%
(97-100)
(87-100)
(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
V12.2
Tissue Culture N= 57
Indeterminants/Unresolved NA
Virus Recovered No. (%)
FluA
2 (3.5%)
0 (0%)
FluB
7 (12%)
8 (14%)
RSV
11 (19%)
1 (2%)
Negative
37 (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 A HMPV B SOIV RSV FLU B Minimum 57.1 59.3 60.9 57.9
25% Percentile
57.8
60.1
58.7
61.45
62.7
Median
58.5
60.7
59.1
61.9
63.5
75% Percentile 59
61.5
59.7
62.5
63.7
Maximum
62.9
60.3
64.3
64.1
Mean
58.47
60.8
59.08
62.05
62.98
Std. Deviation
0.7311
0.9261
0.7714
0.8635
1.389
Std. Error
0.1954
0.1931
0.177
0.1884
0.3187
Lower 95% CI of mean
58.05
60.4
58.71
61.65
62.31
Upper 95% CI of mean
58.89
61.2
59.45
62.44
63.65
Coefficient of variation
1.25%
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 H1N1 2009 – 10 1 TCID 50/ml with Tm 57 - 61

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
Mycoplasma pneumoniae
LOD = 100 copies/ml

37. Multiplex assay
LOD = 10-1 pfu/ml
LOD = 28 copies/ml

38. Optimize Specimen

40. Legionella

41. Optimize primer probe concentration

42. Throughput

43. Nested PCR Runs

44. Reliability Two instruments Software upgrade in April and August
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 Daily Monthly
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
0.0985
P value summary ns
Are means signif. different? (P < 0.05) No
Number of groups 9 F
1.79
R squared
0.2036
ANOVA Table SS df MS
Treatment (between columns)
45.74 8
5.718
Residual (within columns)
178.9 56
3.195
Total
224.7 64
No post tests. P > 0.05

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

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

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 S75. 28th 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. M12. 28th Annual Clinical Virology Symposium