About CLAMP sequencing and vaccine-like PCR for PRRS virus (PRRSV)

Following the PRRS Forum organized by the CRIPA and the PRRS workshop held by the AVIA, we received

Following the PRRS Forum organized by the CRIPA and the PRRS workshop held by the AVIA, we received questions regarding two diagnostic techniques and found it useful to give a brief update.

CLAMP sequencing

When a sample contains a mixture of PRRSV strains, sequencing by the Sanger method is usually able to sequence only one of the strains, the predominant strain in the sample.
CLAMP sequencing can obtain the sequence of a wild strain from a sample containing a vaccine strain that predominates over the wild strain.

It selectively amplifies the ORF5 of the wild strain(s) present in the sample by specifically blocking amplification of the vaccine strain’s ORF5. If the vaccine strain is present in the sample (only one at a time), its ORF5 will not be amplified.

To do this, a short nucleopeptide sequence is used, homologous to a region of the ORF5 specific to the vaccine strain in question (i.e. an ORF5 sequence not found in the wild strains). This sequence specifically blocks amplification of the vaccine strain’s ORF5.

The ORF5 sequence obtained at the end of the PCR belongs to the wild strain(s) and not to the vaccine strain present in the sample.

It should be noted that CLAMP PCRs are less sensitive than the PCRs usually used for sequencing. To work, the Ct value of the wild strain must be < 30. To date, the CLAMP sequencing test is offered exclusively by the Veterinary Diagnostic Laboratory of Iowa State University.

The vaccine-like PCR test

The vaccine-like PCR test is used to determine whether a sample positive in the screening PCR may contain a vaccine strain (MLV, ATP or Fostera) or only a wild strain.
It selectively amplifies regions of the ORF5 specific to the MLV, ATP and Fostera vaccine strains. It then compares the results obtained by the screening PCR, which targets ORF7, or eventually another region of the genome (which varies according to the PCR).

If the Cts obtained in the 2 tests are close, it is concluded that the sample probably contains one of the MLV, ATP or Fostera vaccine strains or a strain derived from them (vaccine-like).

If the Cts are distant, the sample probably does not contain a vaccine strain but one (or more) wild strains.

Note that the sensitivity of the vaccine-like PCR is equivalent to or sometimes better than that of the screening PCR test.

The vaccine-like PCR test (MLV, ATP, Fostera) is offered at Biovet.

Please feel free to contact us for further information.

Christian Savard, Director, Molecular Biology R&D
André Broes, Technical Support Manager

References

  1. Harmon K, Bradner L, Bieber M, Gauger P, Zhang J. Put a CLAMP on it! PCR-based strategy to selectively sequence wild-type PRRSV in vaccinated herds. Proc. Annual meeting AASV. 2021, 50-52
  2. Gauger P, Harmon K. PRRS CLAMP: molecular diagnostic tools to distinguish wild-type and vaccine strains of PRRSV. Proc. Annual meeting AASV. 2021, 371-374
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