Nucleic acid Amplification Assay Development using the BD MAX

Sue C. Kehl, Ph.D. D(ABMM)Associate Professor, PathologyMedical College of Wisconsin

Associate Director, Clinical PathologyTechnical Director, Microbiology and Molecular Infectious Disease

Children’s Hospital of Wisconsin

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

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

Serial dilutions of RSV stock detected with expected melts

Serial dilutions of Influenza A

Serial dilutions of Influenza B stock detected with expected melts

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

0 10 20 30 40 50 60

-6

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-1.4

cycle number

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Optimize primer/probe concentrations

0 10 20 30 40 50 60

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-3.5

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-1.4

5 H1 10^4 6 H1 10^3 7 H1 10^2 8 H1 10^1 9 H1 10^0

cycle number

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)

Protocol optimizationTIME (s) TEMP

UNG 300 40RT 1800 50DENATURE 120 95PCR X 45

‘ DENATURE 15 95‘ ANNEAL 30 56‘ EXTENSION 30 76DENATURE 15 95MELT 40-95

Previous protocolPredicted time: 1.7 hrsActual time: > 2.5 hrs

Altered protocol to decrease timeEliminate UNGDecrease RT timeShorten melt range

Actual time: 2 hours

Multiplex Assay• Prepare Multiplex• Repeat Limit of Detection

RSV

Influenza A

Influenza B

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

RSV Cross Talk

RSV Cross Talk Elimination

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

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 - 400 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.

  Ct value

  Manual easyMAG EZ1 SingleplexBD

MAX

RSV 10 -1 TCID 50/ml 37.6 36.8 35.0 37 36.3INFLUENZA A 10 1

TCID 50/ml 32.4 33.7 31.2 36 36.8INFLUENZA B 10 1

TCID 50/ml 33.0 31.8 34.6 35.3 34.9

Contamination

• High concentration of virus– Tested alternating locations in two runs

• No evidence of contamination or carryover

Threshold• Most of the assay development had used

instrument calculated threshold (-1.6)• Specimens were considered positive if Ct < 40

with an appropriate Tm

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

15 20 25 30 35 40 45

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1 CLINICAL SAMPLE #101 2 CLINICAL SAMPLE #102 3 CLINICAL SAMPLE #103 5 CLINICAL SAMPLE #105

7 CLINICAL SAMPLE #107 8 CLINICAL SAMPLE #108 9 CLINICAL SAMPLE #109 12 NEGATIVE

threshold

cycle number

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-0.02

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0.12

1 CLINICAL SAMPLE #101 2 CLINICAL SAMPLE #102 3 CLINICAL SAMPLE #103 5 CLINICAL SAMPLE #105

7 CLINICAL SAMPLE #107 8 CLINICAL SAMPLE #108 9 CLINICAL SAMPLE #109 12 NEGATIVE

Temperature (C)

-dF

/dT

(V

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Threshold

0 10 20 30 40 50 60

-6

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-3

-2

-1

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-0.940000000000001

1 H3N2 10^0 2 H3N2 10^-1 3 H3N2 10^-2 4 FLU B 10^45 FLU B 10^3 6 FLU B 10^2 7 SKIP WATER 8 FLU B 10^19 FLU B 10^0 10 FLU B 10^-1 11 FLU B 10^-2 12 WATERRaider Cts threshold

cycle number

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40 45 50 55 60 65 70 75 80 85

-0.01

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0.06

FAM Target Melt Simple -dF vs. T1000052, , Monday December 01 09:28:06AM 2008,

1 H3N2 10^0 2 H3N2 10^-1 3 H3N2 10^-24 FLU B 10^4 5 FLU B 10^3 6 FLU B 10^27 SKIP WATER 8 FLU B 10^1 9 FLU B 10^010 FLU B 10^-1 11 FLU B 10^-2 12 WATER

Temperature (C)

-del

ta R

F (

V)

•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

15 20 25 30 35 40 45

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-3.5

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1 H1N1 10^3 2 H1N1 10^2 3 H1N1 10^1 4 H1N1 10^0 5 H1N1 10 -̂1

6 H1N1 10 -̂2 7 H3N2 10^3 8 H3N2 10^2 9 H3N2 10^1 10 H3N2 10^0

11 H3N2 10 -̂1 12 H3N2 10 -̂2 threshold

cycle number

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45 50 55 60 65 70 75 80

-0.02

0

0.02

0.04

0.06

0.08

0.1

1 H1N1 10^3 2 H1N1 10^2 3 H1N1 10^1 4 H1N1 10^0 5 H1N1 10^-1 6 H1N1 10^-2

7 H3N2 10^3 8 H3N2 10^2 9 H3N2 10^1 10 H3N2 10^0 11 H3N2 10^-1 12 H3N2 10^-2

Temperature (C)

-dF

/dT

(V

)

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

• 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

Table 1. Comparison of frozen tissue culture specimens in Multiplex RT-PCR assays on Two color BD MAX

  ProFlu V12.2N= 92* 93Indeterminants or Unresolved 6 7

 Sensitivity

(95% CI)Specificity

(95% CI)Sensitivity

(95% CI)Specificity

(95% CI)

FluA N=1693%

(66-100)100%

(95-100)100% (74-100)

100% (96-100)

FluB N=2180% (56-94)

100% (95-100)

90%*** (70-99)

100% (95-100)

RSV N=2095% (73-100)

95% (87-98)

100% (83-100)

96% (100)

         

Total88% (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.

Table 2. Virus recovery from fresh specimen in tissue culture

  V12.2 Tissue Culture

N= 57 57

Indeterminants/Unresolved 0 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

Precision Ct

• Test known concentration 5 times within a run• Test known concentration over 10 days• Reasonable expectation is + 0.5 log, which

correlates to 5%• Our assay showed coefficient of variation of

2.1 – 2.7%

Precision TmHMPV A HMPV B SOIV RSV FLU B

Minimum 57.1 59.3 57.1 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 59.7 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%

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

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• H1N1 2009 labeled with Cal Red 585

– Document dynamic range– Determine analytical sensitivity– Determine analytical specificity– Determine clinical sensitivity and specificity

• Limit of detection– H1N1 2009 – 10 1 TCID 50/ml with Tm 57 - 61

– H3N2 - 10 0 TCID 50/ml with Tm 50 - 55

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Ch1 (FAM) Average Background(3-15) LOG (delta rxn) vs. cycle number_x000d_SUZANNE, Tuesday January 03 02:14:51PM 2012, , SW 1000060

1 H1 1 2 H1 2 3 H1 3 4 H1 4 5 H1 5 6 H1 6 7 H1 7 8 H1 8

9 H1 9 10 H1 10 11 POS CT H1N1 041411SOIV 12 H3 1 threshold

cycle number

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15 20 25 30 35 40 45

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-3.5

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-0.5

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Ch2 (AP593) Average Background (3-15) LOG(delta rxn) vs. cycle number_x000d_SUZANNE, Tuesday January 03 02:14:51PM 2012, , SW 1000060

1 H1 1 2 H1 2 3 H1 3 4 H1 4 5 H1 5 6 H1 6

7 H1 7 8 H1 8 9 H1 9 10 H1 10 11 POS CT H1N1 041411SOIV 12 H3 1

threshold

cycle number

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-0.02

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0.02

0.04

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0.08

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Ch1 (FAM) Melt ProfileADMIN, Tuesday January 25 01:59:52PM 2011, , SW 1000084

1 H1 1 2 H1 2 3 H1 3 4 H1 4 5 H1 5 6 H1 6 7 H1 7

8 H1 8 9 H1 9 10 H1 10 11 POS CT H1N1 041411SOIV 12 H3 1

Temperature (C)

-dF

/dT

(V

)

45 50 55 60 65 70 75 80

-0.02

1.04083408558608E-17

0.02

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0.1

Ch2 (AP593) Melt ProfileADMIN, Tuesday January 25 01:59:52PM 2011, , SW 1000084

1 H1 1 2 H1 2 3 H1 3 4 H1 4 5 H1 5 6 H1 6 7 H1 7 8 H1 8

9 H1 9 10 H1 10 11 POS CT H1N1 041411SOIV 12 H3 1

Temperature (C)

-dF

/dT

(V

)

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

Mycoplasma pneumoniae

Chlamydophila pneumoniae

LOD = 10 0 pfu/ml

LOD = 100 copies/ml

Multiplex assay

LOD = 10-1 pfu/ml

LOD = 28 copies/ml

Optimize Specimen

Legionella

Optimize primer probe concentration

Throughput

RNA - 12 RNA - 24 DNA - 12 DNA - 24Sample Log-in 2-3 4-6 2-3 4-6Verification 1-2 2-4 1-2 2-4Extraction 75 147 62 90Load cartridge 5 13 6 13PCR 120 120 90 90

Total 3 hrs 25 min 4 hrs 50 min 2 hrs 45 min 3 hrs 25 min

Nested PCR Runs

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 CM0073 - 0%• Lot 4 CM0090 - 0%

Quality Control• New Quantitated virus stocks

– Test 5 times in assay– Determine acceptable Ct range (mean Ct + 2.5 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

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

• 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

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

References• Khanna, M., J. Fan, K. Pehler-Harrington, C. Waters, P. Douglass, J. Stallock, S. Kehl,

K.J. Henrickson. 2005. 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. 2005 Feb;43(2):565-71.

• 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. 2009. Rapid semi-automated subtyping of influenza during the 2009 swine-origin Influenza A H1N1 epidemic in Milwaukee, Wisconson. J Clin Microbiol. 47(9):2779-2786.

• 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. 2009. Comparison of a novel multiplex automated laboratory-developed RT-PCR assay for RSV and Influenza A/B with the ProFLU+ Assay. M20. 25th 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. 2009. 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 58. 25th 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. 2009. 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. M63. 25th Annual Clinical Virology Symposium.

References• Kehl, S.C., S. Goodacre, K. Vaughn, N. Ledeboer, K.J. Henrickson. 2009. Comparison of

the Jaguar System, the Qiagen Biorobot EZ1 and the NucliSens easyMAG for extraction of Enterovirus, RSV and Influenza A. M64. 25th 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. 2010. 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. 2010 12: 74-81; doi:10.2353/jmoldx.2010.090095

• Trost, J.F., E. T. Beck, M. E. Bose, K. Waant, J.Fan, S. Kumar, S. C. Kehl, K. J. Henrickson. 2012. 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. 2012. Fully-automated multiplex Real-Time RT-PCR for identification Of Influenza A, Influenza B, RSV, and hMPV. M12. 28th Annual Clinical Virology Symposium