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BAM 26B Appendices: Multi-laboratory Validation of Hepatitis A Virus Concentration and Detection Protocols

 

BAM Chapter 26B   |   BAM Menu


Appendix A – Single Laboratory Real-time RT-PCR Validation Results

The real time RT-qPCR assay for phase 1A and 1B included a single and multi-laboratory validation. For phase 1A, the SLV of the HAV (ATCC VR-1402) detection assay, three HAV strains were used to establish inclusivity (Appendix A, Table 1) Eleven enteric viruses and five pathogenic enteric bacterial species were used to establish assay exclusivity (Appendix A, table 2). Hepatitis A virus positive and negative human sera samples from CDC were tested using the HAV detection assay and results demonstrated 100% accuracy in detection (Appendix A, table 3).  The amplification efficiency ranged between 97 and 103% (Appendix A, table 4). Acceptable ranges of amplification efficiency are from 90 to 110% (Pfaffl, 2004). There was no inhibition in the assay with the addition of the internal amplification control (IAC) and competitive RNA poliovirus (Appendix A, Table 5). The dynamic range of the assay was 7 logs (Appendix A, Figure 1) and the limit of quantification and limit of detection were 0.11 and 0.001 PFU/ reaction, respectively (Appendix A, Table 6).

Sample Prep

Template for the detection assay consisted of extracted RNA or boiled preps of DNA. QIAmp Viral RNA kits (Qiagen, Carlsbad, CA) was used to extract RNA and DNA samples were heated to 95°C to release the DNA. For the four stages of the MLV, each stage was completed 1 to 2 months apart. Template and PCR reagents were shipped overnight on dry ice and stored at -20°C until analysis.

RNA Template Controls and Clinical Specimens

Viral Nucleic Acid Templates (for inclusivity and exclusivity testing)

RNA was isolated from stock suspensions and diluted for inclusive and exclusive, viruses (Appendix A Tables 1 and 2) and stool samples using the QIAamp Viral RNA Mini Kit (Qiagen) protocol for cell culture. The RNA was eluted from the spin columns with 60 µl AVE elution buffer (provided in kit) and stored at -80 °C until used.

RNA Template for Competitive RNA testing

Poliovirus RNA was isolated from stock suspensions, diluted and added to the HAV RT-qPCR multiplex assay to determine if the presence of additional enteric viral RNA would be competitive with detection of HAV viral RNA. The poliovirus RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) protocol for cell culture. The RNA was eluted from the spin columns with 60 µl AVE elution buffer (provided in kit) and stored at -80 °C until used.

Bacterial Templates (for exclusivity testing)

DNA template were prepared by transferring 1ml of overnight Tryptic Soy Broth culture to a microcentrifuge tube and centrifuge 12,000 × g for 3 min. The supernatant was removed and the pellet completely resuspend in 1 ml 0.85% NaCl. The tube was centrifuge 12,000 × g for 3 min. The supernatant was removed and the pellet was completely re-suspend  in 1 ml sterile water. The tube was place in waterbath or heat block and maintained at 100°C for 10 min. Following boiling the tube was then centrifuge 12,000 × g for 1 minute, and the supernatant was removed to a new microcentrifuge tube. This bacterial extracted was frozen,at -20 °C and served as the appropriate control.

Table 1. Analytical Specificity Studies of the HAV assay - Inclusivity

StrainSource ATCCAve Ct.SDIAC Avg CtSDFrequency
HM175/18f (sub-genotype 1B)VR-140229.080.20820.880.226 of 6
PA21 (sub-genotype IIIA)VR-135723.240.26120.850.336 of 6
PA21 (sub-genotype IIIA)VR-228120.070.35221.2211.086 of 6

Replicates = 6

 

Table 2. Exclusivity testing of organisms that can be associated with fecal contamination

Exclusivity Study w/ LHAV Primer/Probe
StrainSourceResultsFrequency
PoliovirusATCC VR-193Negative6 of 6
AstrovirusHuAst1Negative6 of 6
San Miguel Sea Lion virus serogroup 17Dr. Alvin Smith, Univ. OR, CorvallisNegative6 of 6
RotavirusATCC VR 2018Negative6 of 6
AdenovirusATCC VR-1083Negative6 of 6
Feline CalicivirusATCC VR-2057Negative6 of 6
Human ParaechovirusATCC VR-1063Negative6 of 6
Echovirus 1ATCC VR-1038Negative6 of 6
CoxackievirusATCC VR-1007Negative6 of 6
Norwalk Virus GIHuman StoolNegative6 of 6
Norovirus GIIHuman StoolNegative6 of 6
Escherichia coliATCC 25922Negative6 of 6
Salmonella entericaATCC 9700Negative6 of 6
Shigella sonneiATCC 9290Negative6 of 6
Vibrio choleraATCC 14035Negative6 of 6
Listeria monocytogenesATCC 7646Negative6 of 6

 

Table 3. CDC serum real-time RT-PCR HAV analysis

SAMPLE NUMBERHAV DETECTIONCT VALUEIAC CT VALUE
17000Positive34.6523.63
14000Positive32.2323.16
13516Positive25.6723.14
12010Positive39.8622.74
12009Positive27.1823.42
17500Positive37.4923.53
12144Positive28.7423.03
16000Positive36.2523.33
12121Positive23.9122.82
12113Positive28.6623.65
12101Positive23.2723.15
12112Positive29.2123.58
13518Positive32.4223.32
12319Positive29.7323.61
12399Positive29.7523.11
12320Positive28.2523.18
12312Positive30.6823.19
12323Positive28.5823.33
12322Positive26.8023.34
12305Positive25.1222.87
12330Positive24.2523.10
12385Positive28.2623.19
12316Positive26.0923.21
12346Positive33.8823.18
12363Positive28.0923.24
12325Positive22.3823.22
12364Positive32.3223.21
12359Positive31.7023.12
12326Positive28.1023.60
12302Positive29.0823.58
12313Positive33.2823.28
12352Positive33.7423.40
12303Positive29.0623.32
12304Positive26.8323.55
12306Positive28.9523.53
12329Positive29.4422.75
12330Positive25.1223.35
12307Positive31.4223.29
12331Positive31.3523.15
12345Positive28.5423.12
12003Negative*-23.41
12013Negative*-23.34
19300Negative*-23.23
13517Negative*-23.28

Samples assayed one time due to limited amount of serum provided
*Negative by CDC method

 

Table 4. Intra-Assay Variability: 3- 100 fold dilutions of HAV RNA, 9 replicates per dilution.

 Trial 1Trial 2Trial 3Trial 4Trial 5
Ct*HighMedLowHighMedLowHighMedLowHighMedLowHighMedLow
Mean21.0927.8534.3728.2434.8221.3827.9234.7034.7321.2628.2434.8221.3827.9234.70
SD0.1820.3200.2430.2500.5990.2490.3380.5230.3470.3500.2500.5990.2490.3380.523
SE0.0610.1030.0610.0830.2000.0830.1130.1980.1160.1170.0830.2000.0830.1130.198
Amplification Efficiency100%103%97%97%100%
r20.9980.9980.9970.9950.996
Internal Control ImpactNo Significant Difference
(p= 0.113)
No Significant Difference
(p= 0.415)
No Significant Difference
(p= 0.183)
No Significant Difference
(p= 0.311)
No Significant Difference
(p= 0.939)

*Threshold Set at 10.
Mean Efficiency: 100%; r2= 0.997

Intra-Assay Reproducibility: Five samples of varying low (Ct 33-36) medium (Ct 27-28) and high (Ct 21-22) concentrations were tested in 9 assays on the same run. The results show excellent reproducibility.

Inter-assay reproducibility: The Ct value of the daily positive control was analyzed over a period of 3 days on two different, calibrated, Smart Cycler blocks. The results show consistent reproducibility over the period of time on the same sample.

 

Table 5. Competitor RNA Study HAV and IAC amplification in the presence of competitive RNA (Polio virus; 4 × 104 pfu/rxn); 6 reactions/ level

 HAV Assay w/o IACIAC Ct
 w/ IAC RNA CtSDw/ Polio CtSDw/ HAV CtSDw/ HAV & PolioSD
HAV 2 × 103 pfu/rxn27.060.16426.970.20722.360.12622.280.094
HAV 20 pfu/rxn32.900.32633.420.40822.180.26422.310.278
HAV 0.2 pfu/rxn41.030.76840.721.44022.340.12922.410.193

 

Figure 1. Dynamic Range of Assay: The assay has a dynamic range of 7 logs and a mean efficiency of 99.4%

Figure 1 (Appendix): Figure A1.  Dynamic Range of Assay: The assay has a

 

Figure 2. Dynamic Range Standard Curve

Appendix: Figure 2.  Dynamic Range Standard Curve: Correlation plots

 

Table 6. Limit of Detection (LOD)/Limit of Quantitation (LOQ)- HM175/18f

 Ave. CtSD*Pos/Total
0.11 PFU41.130.5510/10
0.01 PFU42.440.827/10
0.001 PFU43.481.048/10

* standard deviation of positive samples only.

LOQ = 0.11 PFU/rxn
LOD = 0.001 PFU/rxn


Appendix B – Multi-Laboratory Real-time RT-PCR Validation Results

The MLV of the HAV detection assay, phase 1B, consisted of four stages conducted by eight participating FDA laboratories (Appendix B, Table 1). Each of the four stages included a minimum of four laboratories. Microorganisms tested for inclusivity and exclusivity included three strains of HAV, four enteric viruses, and enteric bacteria (Appendix B, Table 2). The inter-laboratory repeatability results demonstrated outliers for two laboratories but these results should be considered analyst error and not a function of the analytical tests performed (Appendix B, Table 11). Overall, results for the MLV demonstrated an accuracy of 99% with a 1% false positive and false negative rate (Appendix B, Table 12). These accuracy rates are within the acceptable limits for Nucleic Acid Technology (NAT) assays.

Sample Prep

Template for the detection assay consisted of extracted RNA using QIAmp Viral RNA kits (Qiagen, Carlsbad, CA). For the four stages of the MLV, each stage was completed 1 to 2 months apart. Template and PCR reagents were shipped overnight on dry ice and stored at -20°C until analysis.

Table 1. Microorganisms Tested used in Multi-Laboratory Validation

Strain
HAV HM 175/18f
HAV PA 21
HAV HAS15
Poliovirus
Astrovirus
San Miguel Sea Lion virus serogroup 17
Norovirus GII
Salmonella enterica

6 replicates/ organism were generally tested but due to
laboratory error only 3 replicates were tested in certain instances.

 

Table 2. Stage #1 Results

Detection of HAV via RT-qPCR
Organism/ StrainLab #1Lab #5Lab #6Lab #7
HAV HM- 1753 of 36 of 66 of 66 of 6
HAV PA 213 of 36 of 66 of 66 of 6
HAV HAS153 of 36 of 66 of 66 of 6
Poliovirus0 of 30 of 60 of 60 of 6
Astrovirus0 of 30 of 60 of 60 of 6
NoV; SMSV-170 of 30 of 60 of 60 of 6
HuNoV GII0 of 30 of 60 of 60 of 6
Salmonella0 of 30 of 60 of 60 of 6

HAV: 63 of 63 reaction positive ; 100% accuracy
Non-HAV: 105 of 105 reactions negative: 100% accuracy

 

Table 3. Stage #2

Detection of HAV via RT-qPCR
Organism/ StrainLab #1Lab #2Lab #3Lab #4Lab #5
HAV HM-1756 of 66 of 66 of 66 of 66 of 6
HAV PA 216 of 66 of 66 of 66 of 66 of 6
HAV HAS155 of 66 of 66 of 66 of 66 of 6
Poliovirus0 of 60 of 60 of 60 of 60 of 6
Astrovirus0 of 60 of 60 of 60 of 60 of 6
NoV; SMSV-170 of 62 of 60 of 60 of 60 of 6
HuNoV GII0 of 60 of 60 of 60 of 60 of 6
Salmonella0 of 60 of 60 of 60 of 60 of 6

HAV: 89 of 90 reaction positive ; 99% accuracy
Non-HAV: 178 of 180 reactions negative: 99% accuracy

 

Table 4. Stage #3

Detection of HAV via RT-qPCR
Organism/ StrainLab #1Lab #4Lab #6Lab #7
HAV HM- 1756 of 66 of 66 of 66 of 6
HAV PA 216 of 66 of 66 of 66 of 6
HAV HAS156 of 66 of 66 of 66 of 6
Poliovirus0 of 60 of 60 of 60 of 6
Astrovirus1 of 60 of 60 of 60 of 6
NoV; SMSV-170 of 60 of 60 of 60 of 6
HuNoV GII0 of 60 of 60 of 60 of 6
Salmonella0 of 60 of 60 of 60 of 6

HAV: 72 of 72 reaction positive ; 100% accuracy
Non-HAV: 179 of 180 reactions negative: 99% accuracy

 

Table 5. Stage #4

Detection of HAV via RT-qPCR
Organism/StrainLab #1Lab #2Lab #5Lab #7Lab #8
HAV HM- 1756 of 66 of 66 of 66 of 66 of 6
HAV PA 216 of 66 of 66 of 66 of 66 of 6
HAV HAS156 of 66 of 66 of 66 of 66 of 6
Poliovirus0 of 60 of 60 of 60 of 60 of 6
Astrovirus0 of 60 of 60 of 60 of 60 of 6
NoV; SMSV-170 of 60 of 60 of 60 of 60 of 6
HuNov GII0 of 60 of 60 of 60 of 60 of 6
Salmonella0 of 60 of 60 of 60 of 60 of 6

HAV: 89 of 90 reaction positive; 99% accuracy
Non-HAV: 149 of 150 reactions negative: 99% accuracy

 

Table 6. Inter-Laboratory Repeatability - Stage #1

HAV StrainLab #1Lab #5Lab #6Lab #7
 MeanSDSEMeanSDSEMeanSDSEMeanSDSE
HM-17521.90.260.1522.30.200.0821.90.210.0921.90.260.11
PA 2121.50.410.2421.60.270.1121.10.190.0821.20.220.09
HAS 15M23.90.470.2724.160.220.0923.60.190.0823.90.190.08

HAV HM 175- 1.3 × 104 pfu/rxn; HAV PA21- 7.3 × 104 pfu/rxn; HAV HAS-15- 6.3 × 104 pfu/rxn
*Results from labs #5 and #7 are significantly different (p= 0.012)

 

Table 7. Inter-Laboratory Repeatability- Stage #2

HAV StrainLab #1Lab #2Lab #3Lab #4Lab #5
 Mean CtSDSEMean CtSDSEMean CtSDSEMean CtSDSEMean CtSDSE
HM-17525.10.210.0927.9*0.770.3125.40.350.1425.30.310.1325.20.160.06
PA 2124.50.200.0827.7*0.180.0724.70.680.2824.20.190.0824.30.360.15
HAS 1526.30.190.0826.30.400.1627.21.210.5026.60.510.2126.20.210.09

HAV HM 175 - 1.3 × 103 pfu/rxn; HAV PA21 - 7.2 × 10 pfu/rxn; HAV HAS-15 - 1.1 × 10 pfu/rxn
*Results from Lab #2 are significantly different than results from the other 4 laboratories (p < 0.05)

 

Table 8. Inter-Laboratory Repeatability - Stage #3  

HAV StrainLab #1Lab #4Lab #6Lab #7
 MeanSDSEMeanSDSEMeanSDSEMeanSDSE
HM-17530.3*0.210.0929.9+0.460.1929.4*+^0.180.0728.7*+^0.440.18
PA 2130.50.440.1829.80.220.0930.00.170.0729.30.240.10
HAS 1530.60.660.2730.80.690.0730.10.170.0729.70.420.17

HAV HM 175 - 1.3 × 101 pfu/rxn; HAV PA21 - 7.3 × 101 pfu/rxn; HAV HAS-15 - 1.1 × 102 pfu/rxn

 

Table 9. Inter-Laboratory Repeatability - Stage #4

HAV StrainLab #1Lab #2Lab #5Lab #7Lab #8
 MeanSDSEMeanSDSEMeanSDSEMeanSDSEMeanSDSE
HM-17528.20.590.2427.70.270.1128.00.420.1724.5*1.39.05528.40.340.14
PA 2128.70.330.1428.70.440.1827.99+0.300.1228.60.230.0928.70.180.08
HAS 1528.60.230.1032.2a1.020.4228.40.220.0928.40.180.0728.90.230.10

HAV HM 175 - 1.3 × 102 pfu/rxn; HAV PA21 - 9.5 × 102 pfu/rxn; HAV HAS-15 - 2.2 × 103 pfu/rxn

*Results from Lab #7 are significantly different than results from the other 4 laboratories (p < 0.05)
+Results from Lab #5 are significantly different than results from the other 4 laboratories (p < 0.05)
aResults from Lab #5 are significantly different than results from the other 4 laboratories (p < 0.05)

 

Table 10. Trial Inter-Laboratory Repeatability - Summary

HAV StrainTrial #1Trial #2Trial #3Trial #4
 Mean CtSDSEMean CtSDSEMean CtSDSEMean CtSDSE
HM-17522.00.280.0625.81.160.2129.60.660.1327.41.630.30
PA 2121.30.340.0825.11.380.2529.90.510.1028.50.400.07
HAS 1523.90.310.0726.50.720.1330.30.650.1328.62.440.45

 

Table 11. Summary of Detection: Inclusivity and Exclusivity 8 labs in total participated in the 4 trials

Organism / StrainAccuracyFalse NegativesFalse Positives
HAV HM-175100%0%-
HAV PA 21100%0%-
HAV HAS1598%2%-
Poliovirus100%-2%
Astrovirus99%-1%
NoV; SMSV-1798%-2%
HuNoV GII100%-0%
Salmonella100%-0%
All HAV Strains99%1% 
All Other Organims/Strains99%-1%

Appendix C – Multi-laboratory Concentration, Extraction, and Detection Results for HAV in Green Onion

In Phase 2, green onions were artificially contaminated with three levels of HAV and an extraction control. In this methods validation, 8 of the 10 participating FDA, CFSAN, or FERN laboratories produce acceptable data consistent with the Office of Foods guidelines for level 3 validations. The overall detection frequency of HAV from green onion spikes were 97% and 75% for the 50 pfu and 5pfu/g samples respectively (Appendix C, Table 2).

Sample Prep

Green onions were purchased from a retail market. The samples were cut in 2" and 5" segments and placed into Whirl-pak® bags. Samples were spiked and held at 4°C for 3 days prior to shipment. Twenty samples, in triplicate, were prepared and shipped in coolers with ice bricks to the participating laboratories by CFSAN’s Moffett Center Institute of Food Safety and Health. Sample analysis was begun within 24 hours of receipt.

Virus inoculum

HAV inoculum used for seeding was the vaccine strain (HAV175) propagated in house utilizing FrHK cell line. Murine norovirus (MNV) used for seeding was murine norovirus-1 propagated in house using RAW 264.7 cell line. Three samples levels were used for validation; Low (5 PFU/g of HAV), High (50 PFU/g of HAV) and uninoculated. MNV was inoculated in all samples at 4 × 102/g RT-PCR units.

Table 1. Laboratory data from detection of HAV in green onion

 Laboratory #
Sample #Key12345678910
1-----+ fp-----
2+, L++- fn+++++++
3+, L- fn++- fn+++- fn- fn+
4+, H+++- fn++++++
5+, H+++- fn++++++
6-----+ fp-----
7+ ,L- fn+++++- fn- fn- fn- fn
8+, H++++++++-+
9+, H++++++++error+
10-----+ fp---error+
11+, H++++++++error+
12+, L++- fn++++- fnerror+
13+, L+++- fn+++- fnerror- fn
14+, L+++- fn+++-error- fn
15+, H++++++++error+
16+, H++++++++error+
17-----------
18+, L+++- fn++++- fn- fn
19+, L++- fn- fn++++- fn+
20+, H++++++++++
Positives+++++++++++
Negatives-----------

Red or fp denotes false positives/lab data not valid/not reported
Yellow or fn denotes false negatives
Pink or error denotes data not valid/not reported/instrument error

 

Table 2. Detection Frequencies of HAV

5 PFU/g HAV- Low Inoculum75%
50 PFU/g HAV- High Inoculum97%
0 PFU/g Negative Controla8%
Real-time RT-PCR Controls
False negative0%
False positive RT-PCR controls0%

a results derived from one laboratory; likely cross contamination.

 

Table 3. Percentage of samples with expected results for each participating laboratory

# correct ÷ # samples; % correct
 HighLowNegatives>RT-PCR Controls
Lab #18 of 8; 100%6 of 8; 75%4 of 4; 100%
Lab #28 of 8; 100%8 of 8; 100%4 of 4; 100%
Lab #38 of 8; 100%5 of 8; 63%4 of 4; 100%
Lab #46 of 8; 75%3 of 8; 38%4 of 4; 100%
Lab #58 of 8; 100%8 of 8; 100%1 of 4; 25% (fp)
Lab #68 of 8; 100%8 of 8; 100%4 of 4; 100%
Lab #78 of 8; 100%7 of 8; 88%4 of 4; 100%
Lab #88 of 8; 100%3 of 8; 38%4 of 4; 100%
Lab #93 of 4; 75% (nv)1 of 4; 25% (nv)3 of 3; 100% (nv)
Lab #108 of 8; 100%4 of 8; 50%4 of 4; 100%

Red or fp denotes false positives/lab data not valid/not reported
Pink or nv denotes data not valid/not reported

 

Conclusion:

The MLV of the HAV concentration and detection protocols have demonstrated the sensitivity and specificity of this assay this is acceptable for analysis of regulatory green onions. The results show the assay is sensitive, reproducible and robust and has established the "Fitness of Purpose" for concentration and detection of HAV. The sensitivity of the real-time RT-qPCR assay is 99% with a false positive and false negative rate of 1%. The detection frequency of the concentration assay was 97% for the high inoculum and 75% for the low inoculum. HAV has not been isolated or detected in any food samples implicated in illnesses since 2005, although several HAV associated outbreaks have occurred. In 2005, HAV was detected in shellfish associated in a multistate outbreak and the detection protocol presented in this validation successfully detected HAV in shellfish extract. The limit of detect for this validated concentration and detection protocol is one to five PFU/g. This is two times lower than a recently published protocol and the European Committee for Standardization protocol (Martin-Latil et al., 2012, EFSA, 2012). Our conclusion is that the HAV concentration and detection protocols can be used as a diagnostic screening method for the concentration of HAV in green onion and detection of HAV in RNA preparations obtained from any food matrix. These assays are ready to be incorporated into the Bacteriological Analytical Manual and ongoing Office of Regulatory Affairs Field Assignments.


Appendix D: – Buffer Solution and Extraction Reagents Recipes

 

  1. Glycine/NaCl Buffer (0.75 M Glycine, 0.15 M NaCl, pH 7.6) (R98)

    NaCl8.8 g
    Glycine (Sigma G-7126 or equivalent)56.3 g
    Distilled Water800 ml H20
    q.s. with distilled water to 1 liter
    pH to 7.6
    Autoclave and store at 4 °C

  2. Primer TE (10mM Tris, 0.1mM EDTA, pH 8.0) (R99)

    1M Tris pH 8.0100µl
    0.05M EDTA20µl
    PCR-grade water(Dnase/Rnase free)9.88ml

  3. ALV Buffer and AVE/Carrier RNA mix

    Volumes of Buffer AVL and carrier RNA–Buffer AVE mix required for the QIAamp *store remaining carrier RNA in 30 µl aliquots at -20 °C.

    No.
    samples
    Vol. Buffer
    AVL (ml)
    Vol. carrier
    RNA–AVE (µl)
    No.
    samples
    Vol. Buffer
    AVL (ml)
    Vol. carrier
    RNA–AVE (µl)
    10.565.6137.2872.8
    21.1211.2147.8478.4
    31.6816.8158.4084.0
    42.2422.4168.9689.6
    52.8028.0179.5295.2
    63.3633.61810.08100.8
    73.9239.21910.64106.4
    84.4844.82011.20112.0
    95.0450.42111.76117.6
    105.6056.02212.32123.2
    116.1661.62312.88128.8
    126.7267.22413.44134.4

Appendix E – Inclusivity/Exclusivity Requirements for BAM Methods

April 19, 2011

Note to Method Developers

The purpose of the inclusivity/exclusivity requirements listed below is to ensure that BAM methods are both sensitive enough and specific enough to be reliably used for the analysis of regulatory food samples. That being said, it is understood that it is not always possible to meet the inclusivity/exclusivity requirements listed herein. For example, only limited numbers of strains may be available for emerging pathogens, as well as for certain viruses and parasites. In these cases, originating laboratories should make a good faith effort to test their methods with the maximum number of available strains. The BAM Council will work with method developers to develop achievable goals on a case-by-case basis when the developers are unable to comply with the requirements of this document.

  1. Bacteria for Both Qualitative and Quantitative Methods*

    The choice of inclusivity strains should reflect the genetic, serological, and/or biochemical diversity of the organisms involved, as well as other factors such as virulence, frequency of occurrence and availability. For all organisms, other than Salmonella spp, a minimum of 30 strains of the target analyte(s) are required for inclusivity testing. For Salmonella methods, the number of target organisms is increased to a minimum of 100 serotypes. These serotypes should reflect the genotypic diversity of the organism and should include all of the subspecies as well as the majority of somatic groups found within the genus Salmonella.

    The choice of exclusivity strains should reflect closely related, potentially cross-reactive organisms. Other factors such as virulence, frequency of occurrence and availability should be considered. Select at least 30 strains of potentially competitive organisms.

    Species/strains specified for use must be traceable to the source. The source and origin of each species/strain should be documented.

  2. Total Viable Count Methods

    There are no inclusivity/exclusivity requirements for Total Viable Count methods.

  3. RNA Foodborne Virus Methods*

    This validation scheme is intended for developers of PCR-based assays to be used to confirm the identity or exclusion of isolated strains including nucleic acid. This validation scheme is for both conventional and real time PCR assays. If validating a real time assay, the platform must be specified. It is strongly recommended that the assay be validated on two to three other platforms.

    The criteria necessary to determine four levels of validation for qualitative PCR assays for viruses are the following:

    Inclusivity and exclusivity - Inclusivity is the ability of the method to detect a wide range of target viral strains by their genetic composition. Exclusivity is the ability of the method to distinguish the target virus from similar and genetically distinct non-target viruses. Strains used for inclusivity and exclusivity tests must be from foodborne viruses, include some cultures of known identity from recognized culture collections.

    It is expected from the originating laboratory that all primer and/or probe sequences would initially be screened for uniqueness by searching a viral genomic database for homology. It is recommended that a BLAST search be performed against the GENBANK viral database and equivalent data bases (ex/ Calicinet).

    Preparation of lysates for RT-PCR template (see Appendix One)

    Template should be prepared from clinical specimens, (stool, vomitus, sera) or from cell culture lysates.

    In the overall PCR-based assay, positive and negative control strains and reactions should be clearly noted. The use of internal amplification controls for real-time PCR assays is required, and preferred for gel-based PCR.

    Level of Sensitivity - The level of sensitivity should be considerate of the number of target cells. A comparable amount of target analyte should be used for both inclusivity and exclusivity.

    Analyte levels -The inoculum level should be adjusted to achieve fractional recovery. Fractional recovery is defined as an inoculum level that will yield 25-75% positive results with at least one of the methods being compared. This is necessary for statistical comparisons between methods. This analyte level may be determined empirically by using more than one inoculum level. A set of uninoculated negative controls are also included.

    Dynamic Range - The number of log dilutions of the analyte that can be detected.

    Level of Sensitivity - The level of sensitivity can be expressed as either the least number of intact target virus (i.e. pfu, TCID50, that can be amplified or from target nucleic acid (i.e. copies).

    Addition of competitor strains - the added strain serves to more closely simulate conditions found in nature. In some circumstances the normal background flora serves this purpose.

    Comparison to reference culture method - Where possible, the method being validated is tested alongside an existing reference culture method.

     

    Four Categories of Validation

    Category One
    This category has a minimal level of validation, with all the work done by the originating laboratory. The inclusivity and exclusivity has been tested, by the indicated strains. The expectation would be for the originating lab to continue to conduct further testing to eventually elevate the method to an increased category of validation.

    Category Two
    This is a more robust study, with increased confidence in the results. The originating lab has done a more comprehensive initial study, with inclusivity/exclusivity strains. Two other independent laboratories have participated in the collaborative study in addition to the originating laboratory. In addition, the originating laboratory will compare the PCR-based method to another reference identification method, (e.g. serological or conventional tissue culture).

    Category Three
    At this category of validation, most of the criteria followed by the originating lab are at the AOAC level, including inclusivity/exclusivity, analyte levels, competitor strains, and comparison to existing method when available. The additional collaborating labs follow many of the criteria of an AOAC collaborative study. The main difference is that Category Three validation employs two additional collaborating labs instead of the ten used by AOAC.

    At Category Three validation methods are suitable for field use, with appropriate concurrence from the Division of Field Science. This level of validation is necessary for submission to and evaluation by the BAM Council.

    Category Four
    This validation category has higher and more stringent criteria than Category Three. The confidence that the PCR-based assay is robust, accurate, and efficient is high. This validation category has criteria equivalent to the AOAC Collaborative Study, or has undergone an AOAC collaborative study.

 

Validation Scheme for RNA Food-Borne Viruses Qualitative PCR Methods – Originating Laboratory

Originating Laboratory StudyCategory One:
Urgent usage
Category Two:
Independent lab validation
Category Three:
Multiple lab collaborative
Category Four:
Replicates per straina1366
Comparison to recognized methodNoYes, if availableYes, if availableYes, if available

a If no validated PCR-based method is available then a biochemical and/or serological method will be used.

 

Inclusivity Panel for Validation

TargetCategory OneCategory TwoCategory ThreeCategory Four
Norovirus1 Strain Genogroup I
1 Strain Genogroup II
2 Strains – Genogroup I
5 Strains – Genogroup II
5 Strains – Genogroup I
10 Strains – Genogroup II
10 Strains – Genogroup I
20 Strains – Genogroup II
Hepatitis AHM175/18f
ATCC #VR-1402
5 Strainsa10 Strainsb20 Strainsb
EnterovirusPoliovirus 1 (attenuated)
ATCC #VR-1562
5 Strainsc15 Strainsd30 Strainsd

 

Norovirus Panels:
Currently NoV or nucleic acid from NoV are commercially available. Robustness of panel will be established on a case by case since it will be dependent upon availability of clinical isolates for testing. However, this panel should include strain that have been mostly commonly associated with foodborne illnesses that have occurred in the U.S. and reported to the CDC in the past 3 years. Representative strains must from Genogroup I and II as described above.

Hepatitis A Panels: Category Two:
aShould Include the Following Strains:HM175/18f (subgenotype 1B);ATCC #VR-1402
 HAS-15 (subgenotype 1A);ATCC #VR-2281
Hepatitis A Panels: Categories Three and Four; Enterovirus Panels: Category Two: and categrories three and four:
bShould Include the Following Strains:HM175/18f (subgenotype 1B);ATCC #VR-1402
 HAS-15 (subgenotype 1A);ATCC #VR-2281
 LSH/SATCC #VR-2266
 PA219 (subgenotype IIIA);ATCC #VR-1357
 
Enterovirus Panels: Category Two:
cShould Include the Following Strains:Poliovirus 1 (attenuated);ATCC #VR-1562
 Coxsackievirus A3;ATCC #VR-1007
 Echovirus 1;ATCC #VR-1038
Enterovirus Panels: Categories Three and Four:
dShould Include the Following Strains:Poliovirus 1 (attenuated);ATCC #VR-1562
 Poliovirus 3 (attenuated);ATCC #VR-63
 Coxsackievirus A3;ATCC #VR-1007
 Echovirus 1;ATCC #VR-1038
 Echovirus 21;ATCC #VR-51

Inclusivity Panel was selected in-part due their availability for purchase from a commercial entity (ATCC).

 

Exclusivity Panel for Validation

TargetCategory OneCategory TwoCategory ThreeCategory Four>
Norovirus10 strainsa20 strainsb30 strainsb30 strainsb
Hepatitis A10 strainsc20 strainsd30 strainsd30 strainsd
Enterovirus10 strainse20 strainsf30 strainsf30 strainsf

 

 

Norovirus Panels: Category One:
aMust include Panel A:HM175/18f (subgenotype 1B);ATCC #VR-1402
(or equivalent)
 Poliovirus (attenuated)ATCC #VR-1562
(or equivalent)
 Feline calicivirusATCC #VR-2057
 Murine calicivirus 
Norovirus Panels: Categories Two, Three and Four:
bMust include Panel A representatives plus: Panel B:HAV; (subgenotype 1A)ATCC #VR-2281
(or equivalent)
 Coxsackievirus A3;ATCC #VR-1007
(or equivalent)
 Echovirus 1;ATCC #VR-1038
(or equivalent)
 Rotavirus;ATCC #VR-2018
(or equivalent)
 Astrovirus 
 San Miguel Sea lion virus (if available) 
 Escherichia coli (1) 
 Salmonella sp. (1) 
 Shigella sp, 
 Vibrio sp (1) 
 Listeria sp. (1) 
 
Hepatitis A Panels: Category One:
cMust include: Panel C:norovirus genogroup I  
 norovirus genogroup II 
 Poliovirus 1 (attenuated);ATCC #VR-1562
(or equivalent)
 Coxsackievirus A3;ATCC #VR-1007
(or equivalent)
Hepatitis A Panels: Categories Two, Three and Four
dMust include Panel C representatives plus Panel D:Echovirus 1;ATCC #VR-1038
(or equivalent)
 Rotavirus; ATCC #VR-2018
(or equivalent) 
 Feline calicivirus ATCC #VR-2057
 Astrovirus  
 Escherichia coli (1)  
 Shigella sp.(1)  
 Vibrio sp. (1) 
 Listeria sp. (1) 
 
Enterovirus Panels: Category One
eMust include Panel E:norovirus genogroup I  
 norovirus genogroup II  
 HM175/18f (subgenotype 1B); ATCC #VR-1402
(or equivalent) 
Enterovirus Panels: Categories Two, Three and Four
fMust include: Panel E representatives plus - Panel F:HAV; (subgenotype 1A) ATCC #VR-2281
(or equivalent) 
 Rotavirus;ATCC #VR-2018
(or equivalent)
 Feline calicivirus ATCC #VR-2057
 Escherichia coli (1) 
 Salmonella sp. (1)  
 Shigella sp.  
 Vibrio sp. (1)  
 Listeria sp. (1)  

Consideration of strains used for inclusivity and exclusivity testing will be dependent upon availability from commercial entities and the US CDC

 

Validation Scheme for RNA Food-Borne Viruses
Qualitative PCR Methods – Collaborative Study

Collaborative StudyCategory One:
Urgent usage
Category Two:
Independent lab validation
Category Three:
Multiple lab collaborative
Category Four:
# of laboratories providing usable dataan/a2510
Replicates per strainn/a366
Comparison to Recognized Methodbn/aYes, if availableYes, if availableYes, if available

a labs providing data are required to run study on same PCR platform
b If no validated PCR-based method is available then a biochemical and/or serological method will be used.

 

PCR-based Assay Validation Scheme for bacteria

 This validation scheme is intended for developers of PCR-based assays to be used to confirm the identity or exclusion of isolated colonies.

This validation scheme is for both conventional and real time PCR assays. If validating a real time assay, the platform and chemistry must be specified. It is strongly recommended that a real time assay be validated on two to three other platforms.

The criteria necessary to determine four levels of validation for qualitative PCR assays for bacteria are the following:

Inclusivity and exclusivity - the Inclusivity/exclusivity requirements in section A above apply here.  

It is expected from the originating laboratory that all primer and/or probe sequences would initially be screened for uniqueness by searching a bacterial genomic database for homology. It is recommended that a BLAST search be performed against the GENBANK bacterial database.

Method to prepare lysates for PCR template: Transfer ¼ to ½ of the colony to 150 µl dH2O contained in a 1.5 ml centrifuge tube. Boil the suspended cells for 5 min, chill in ice and either store at 4 or -20°C. From an overnight broth culture, centrifuge at 5,000 × g 1.0 ml in a 1.5 ml tube. Remove supernatant, suspend cell material in 250 µl dH20 and boil, chill as above. 

NB: Target gene(s) and control (positive and negative). In regards to target gene(s), the only acceptable target(s), whether to a virulence factor or taxonomic identifier (e.g. 16S DNA), should definitively demonstrate specificity for that particular pathogen. In addition, in the overall PCR-based assay, positive and negative control strains and reactions should be clearly noted. The use of internal amplification controls for real-time PCR assays are not required.

NB: The amount of template, whether using bacterial cells or purified nucleic acid, should be comparable and at a level that will yield an amplicon after amplification by PCR. A comparable amount of target analyte should be used for both inclusivity and exclusivity studies.

Comparison to recognized method - When possible, the originating laboratory will compare the PCR-based method to another PCR-based reference identification method. If a PCR-based method is not available, use a bacteriological, biochemical, and/or serological reference method.


Appendix F – Template Preparation

Nucleic Acid Template

In an effort to minimize the cost and reducing the complexity of shipping infectious microorganisms, including bacteria and viruses, should be sent as purified nucleic acid. Sending samples in this manner should assist in maintaining sample integrity and sample uniformity between collaborating laboratories.

Nucleic acid of testing microorganisms should be derived, if possible, from propagated pure cultures of each organism to initial concentrations adequate for thorough testing to be performed. For suitable nucleic acid concentrations the test strains should be grown to a density of > 1 ×106/ml. Instances where viruses must be propagated in mammalian cells, nucleic acid from uninoculated culture cells must also be tested by the proposed method to assure no cross reactivity. Test organisms that are difficult or are unable to be propagated tested by convention tissue culture techniques (i.e. HAV, human norovirus) will likely be derived from clinical specimens (i.e. blood serum, vomitus, feces).

Nucleic acids derived from bacteria and viruses used for validation testing can be derived in two general manners;

  1. For bacterial cultures only- Boil bacterial culture for 5 min and then immediately chill on ice; centrifuge for 5 min at 5000 × g @ 4°C; aspirate supernatant and store at -80°C in suitable size aliquots.
  2. For Bacterial and Viral cultures- Use commercially available nucleic acid extraction kits. These kits generally use a chaotropic solution and silica based binding columns. Kits are available that are specialized for binding DNA or RNA and should be according to the type of nucleic acid that will be tested.

Kits that have been found suitable for this are manufactured by but not limited to include Ambion, Invitrogen, Qiagen, Roche Applied Science, and Zymo. This list should not be construed as an endorsement of these companies or their products. Purified nucleic acid should be stored at -80°C whenever possible and that RNA templates should be supplement with an RNAse inhibitor to reduce the possible degradation. 


Appendix G – Murine Norovirus Extraction Control Preparation

Sample Extraction Control Murine Norovirus (MNV) Preparation

Materials: Diluent - PBS (tissue culture grade); Formulation found in Solutions Work Sheet of the Extraction protocol

  1. Murine Norovirus - Propagated in RAW cells at FDA’s Gulf Coast Seafood Laboratory. Last titer concentration: 7.7 × 106 RT-PCR units/ml (≈ 7.7 × 104 pfu/ml): titer may vary – see below
  2. Defrost thoroughly MNV stock solution and vortex for 5 sec.
  3. Spin for 2 seconds in a microcentrifuge to bring solution to bottom of tube.
  4. Prepare a 1:10 dilution blank for MNV with 900 µl of PBS.
  5. Add 100 ul of MNV stock solution to 900 µl dilution blank and vortex for 5 sec.
  6. Spin for 2 seconds in a microcentrifuge to bring solution to bottom of tube

    Note: This constitutes the (-1) dilution and results in a final concentration of 7.7 × 105 RT-PCR units/ml.

    Note: aliquots can be frozen for future seeding but titer will diminish after multiple freeze-thaw cycles.

A 100 µl aliquot of this (-1) dilution contains approximately 7.7 × 104 RT-PCR units. This aliquot serves as the extraction control mentioned in step 2 (concentration and extraction work instruction details). For this titer, MNV is added at a final concentration of approximately 1540 RT-PCR units/gram for 50 g of the food matrix (green onion, berries, shellfish etc.). Please be mindful that Murine norovirus is added to samples to determine if extractions were performed correctly and not to establish the sensitivity of the assay. This is a qualitative assay only.

Note: Should a different source of MNV be obtained or was re-propagated it will be necessary to titer this aliquot prior to using this material as an extraction control.

 

The MLV of the HAV detection assay, phase 1B, consisted of four stages conducted by eight participating FDA laboratories (Appendix B, Table 1). Each of the four stages included a minimum of four laboratories. Microorganisms tested for inclusivity and exclusivity included three strains of HAV, four enteric viruses, and enteric bacteria (Appendix B, Table 2). The inter-laboratory repeatability results demonstrated outliers for two laboratories but these results should be considered analyst error and not a function of the analytical tests performed (Appendix B, Table 11). Overall, results for the MLV demonstrated an accuracy of 99% with a 1% false positive and false negative rate (Appendix B, Table 12). These accuracy rates are within the acceptable limits for Nucleic Acid Technology (NAT) assays.
 
Sample Prep
 
Template for the detection assay consisted of extracted RNA using QIAmp Viral RNA kits (Qiagen, Carlsbad, CA). For the four stages of the MLV, each stage was completed 1 to 2 months apart. Template and PCR reagents were shipped overnight on dry ice and stored at -20°C until analysis.

In Phase 2, green onions were artificially contaminated with three levels of HAV and an extraction control. In this methods validation, 8 of the 10 participating FDA, CFSAN, or FERN laboratories produce acceptable data consistent with the Office of Foods guidelines for level 3 validations. The overall detection frequency of HAV from green onion spikes were 97% and 75% for the 50 pfu and 5pfu/g samples respectively (Appendix C, Table 2).

 
Sample Prep
 
Green onions were purchased from a retail market. The samples were cut in 2” and 5” segments and placed into Whirl-pak® bags. Samples were spiked and held at 4°C for 3 days prior to shipment. Twenty samples, in triplicate, were prepared and shipped in coolers with ice bricks to the participating laboratories by CFSAN’s Moffett Center Institute of Food Safety and Health. Sample analysis was begun within 24 hours of receipt.
 
Virus inoculum
 
HAV inoculum used for seeding was the vaccine strain (HAV175) propagated in house utilizing FrHK cell line. Murine norovirus (MNV) used for seeding was murine norovirus-1 propagated in house using RAW 264.7 cell line. Three samples levels were used for validation; Low (5 PFU/g of HAV), High (50 PFU/g of HAV) and uninoculated. MNV was inoculated in all samples at 4 x 102/g RT-PCR units.