National Veterinary Services Laboratories 

Avian Paramyxovirus-1 Real-Time RT-PCR

Proficiency Test Summary 

1. Composition of proficiency test panel: Each panel consists of ten, 1.25 ml samples of betapropriolactone (BPL) inactivated Avian Paramyxovirus-1 (LaSota and Texas G.B.) and avian 

influenza 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 10.

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 testing 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 APMV-1 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 paramyxovirus-1 proficiency test 

should be conducted using the real time RT-PCR assay as outlined in the current version of SOPAV-1505 and the associated instrument-specific documents. All samples are screened using the 

APMV-1 Matrix primer and probe set, and Matrix positives are tested by the vNDV pathotyping 

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 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 (identification of negatives, Matrix 

positives, and vNDV positives).

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

negative samples. Results are compiled and sent to the NVSL statistician for statistical analysis. 

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 both the Matrix and vNDV assays is necessary for approval to conduct testing 

with the APMV-1 rRT-PCR assay. Successful completion implies the individual is proficient on 

all test methods outlined and described in the current version of SOP-AV-1505 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 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 results in that 

participant not being allowed to conduct testing by the APMV-1 rRT-PCR assay. If all 

personnel form a laboratory fail, the laboratory is not approved for testing for that disease. 

Those laboratories that show repeated failed 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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