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Mismatched Primers Create Vietnam and Korea  False Negatives

Recombinomics Commentary
May 11, 2005

>> Primers are tiny strands of synthetic nucleic acid used in PCR or polymerase chain reaction testing. If they are a perfect match for the influenza strain, primers used in flu tests should bind to the RNA of the virus.

Kobasa and lab technician Laura Hart spent several weeks at the National Institute for Hygiene and Epidemiology in Hanoi, sharing diagnostic expertise and helping Vietnamese scientists assess their testing proficiency.

That is when the problem with the primers came to light.

"There've been enough changes in the viruses between last year and this year that we found some of our PCR primers did not work that well," says Kobasa, a researcher in the division of respiratory viruses.<<

The above comments cite primer problems at the National Institute for Hygiene and Epidemiology in Hanoi, which is where H5N1 bird flu testing for northern Vietnam is done.  Previously, false negatives were identified at the Pasteur Institute in Ho Chi Minh City, where testing for H5N1 in southern Vietnam is done.  The generation of the false negatives is due in part to mismatches in primers.

Similar problems have arisen in attempts to verify WSN/33 sequences in pigs in Korea.  Those PCR tests are difficult because the pigs are infected with 2 or 3 viruses (H1N1 WSN/33, H1N2 reassortant found in pigs in the United States, H9N2 Korea avian isolates.

Primers are short pieces of genetic information generated by the investigator to initiate (or prime) the PCR (Polymerase Chain Reaction) assay.  The primers are made in pairs.  One primer pairs up with one strand of the genetic RNA (influenza is an RNA virus where its genes are made of RNA instead of DNA) and the other primer binds to the opposite strand about 600 bp downstream.  The viral RNA between the two primers is then copied and amplified in the PCR assay.  The amplified information is then sequenced to get the genetic information for the RNA between the two primers.

If the primers don't match the viral RNA, yields will be lower, or with enough mismatching, the data will be negative (both primers need to bind for the assay to work).  In clinical samples, where the amount of initial viral RNA may be low, the lowered sensitivity of the assay may generate a false negative.  In cases where more than one virus has infected the same host, such as the pigs in Korea, if only one primer set is used it could generate one sequence and miss the other two (creating the false impression that the assay worked, and although it technically worked for one virus, it created false negatives for the other two).  If the primer pair binds to two different sequences, the mixed signals can be generated where data for both sequences would be present, and generate a sequence that is more difficult to read.

The problem of false negatives due to poorly matched primers can generate false negatives for clinical samples, and can reduce the detection of new sequences, which are necessary to generate more specific primers. 

H5N1 in Vietnam and WSN/33 in Korea however, do not need lab detection to continue to evolve and infect, which is why exclusion of patients based on negative PCR tests is very dangerous to the world's health.

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