National Veterinary Services Laboratories 

Avian Influenza (AI)/Swine Influenza (SIV) Real-Time RT-PCR

Proficiency Test Summary 

1. Composition of proficiency test panel: Each panel consists of fifteen, 1.25 ml samples of 

beta-propriolactone (BPL) inactivated avian influenza (low pathogenic AI subtypes H1, H5 and 

H7), swine influenza (pandemic H1N1)), and avian paramyxovirus type 1 viruses. The panel 

contains blind duplicates, serial dilutions, and negative extraction controls (Tris-Buffered 

Tryptose Broth (TBTB)). The samples are coded with numbers 1 through 15.

2. Cost of proficiency test: Click here to view current NVSL Catalog of Reagents. Additional 

information on diagnostic reagents, including ordering, payments, shipping details and fees, is 

located at www.aphis.usda.gov/nvsl. Then click on the Reagents and Proficiency Tests link.

No cost to approved NAHLN laboratories.

3. Storage conditions: -20 C or lower in a non-frost free freezer.

4. Sample preparation/selection criteria: A limit of detection assay is performed on each 

panel member. Samples with high, medium, and low concentrations of the target analyte are 

chosen for incorporation into the panel.

5. Panel quality control: Limit of detection is conducted to determine high, medium, and low 

analyte specimens. Following selection of the specimens, testing is conducted to determine the 

expected cycle threshold (Ct) for each specimen with real-time instrumentation that is described 

within the current version of SOP-AV-1520 (Smart Cycler), SOP-AV-1521 (AB 7500 Fast), 

SOP-AV-1523 (AB 7900 HT), and SOP-AV-1524 (Roche LightCycler 480).

6. Timing of the proficiency test distribution and data collection: The AI rRT-PCR 

proficiency tests are administered annually in January. Results are collected 4 weeks after 

distribution. 

7. Test method: Performance and interpretation of the avian influenza proficiency test should 

be conducted using the real time RT-PCR assay as outlined in the current versions of SOP-AV1510 (AI) and SOP-BPA-9034 and the associated instrument-specific documents. For AIV 

testing, the samples are screened using the AI matrix primer and probe set, and matrix positives 

are tested by the H5 and H7 subtype assays. For SIV testing, the samples are screened using the 

SIV matrix primer and probe set, and matrix positives are tested by the pandemic N1 subtype 

assay.

8. Submitting test results: Participants are required to have data submitted for scoring no more 

than four (4) weeks after panel distribution. Results are reported to the administering laboratory, 

the Diagnostic Virology Laboratory (DVL), at the NVSL, by fax or e-mail. Results for all 

laboratories are kept at the testing laboratory office.

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9. Scoring of individual panel samples: For each sample, a participant is considered as 

passing if the unknown samples are identified correctly (e.g., identification of negatives, matrix 

positives, H5 positives, H7 positives, and pN1 positives).

10. Laboratory pass/fail criteria: The final score is based on the identification of positive and 

negative samples. Results are compiled and analyzed by statistical methods. The minimum 

passing score is 90%. Successful completion of the matrix, H5 and H7 assays or the SIV matrix 

and pN1 assays are necessary for approval to conduct testing with the AI or SIV rRT-PCR assay, 

respectively. Successful completion implies the individual is proficient on all test methods 

outlined and described in the current version of SOP-AV-1510 or SOP-BPA-9034 and the 

associated documents.

11. Reporting laboratory test scores: Results for each laboratory are reported only to the 

respective laboratory director. The director is asked to share the results with each individual 

participant. The Final Report on the Proficiency Panel Test is compiled and sent to laboratory 

directors along with a letter of approval or failure within 60-90 days of the receipt of 

participants’ results. Approval letters are mailed separate from non-approval letters. A copy of 

the results for all National Animal Health Laboratory Network (NAHLN) laboratories is sent to 

the NAHLN Program Office.

12. Remedial actions required for failing laboratories: Individual participants from a 

laboratory must pass all parts of the proficiency test, as described in section 10, in order for that 

person to perform either the AI or SIV rRT-PCR assays. Individual personnel from a laboratory

that do not successfully complete the proficiency test on the first attempt are given a retest.

Failure to successfully complete the proficiency test on the retest means that participant is not 

allowed to conduct testing by the rRT-PCR assay. If all personnel from a laboratory fail, the 

laboratory is not approved for testing for that disease. Those laboratories that show repeated fail 

attempts are encouraged to contact the administering laboratory for discussion of potential areas 

of concern. These laboratories are asked to test again in the next round of testing. If requested 

by the laboratory, additional training samples may be provided to the laboratory for practice 

purposes. Additional training may be provided if requested by the NAHLN laboratory, by the 

NVSL and/or by the NAHLN Program Office.

13. Special requirements: Only laboratory personnel who have successfully completed an 

approved Avian Influenza/Newcastle Disease Virus rRT-PCR training course are eligible to

participate in this proficiency test. Training may be administered by the NVSL or through the 

Train-the-Trainer program. Training received at a laboratory other than the NVSL must be 

approved by the NAHLN Program Office in conjunction with NVSL. Participating NAHLN 

laboratories must comply with provisions outlined in the NAHLN Laboratory Qualification 

Checklist for Membership of a Veterinary Diagnostic Laboratory.

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