A TaqMan-MGB real-time PCR for discriminating between MS-H-live vaccine and fieldstrains.

Abstract

(MS) is an important pathogen in the poultry industry and has caused significant economic losses. Worldwide, the use of live attenuated vaccine for the MS-H strain has increased to prevent MS infection. However, there is no test available to discriminate the MS-H vaccine strains from the MS strains that are causing field infection. In this study, a TaqMan-MGB real-time PCR method (qPCR) was established, validated, and evaluated to discriminate between MS-H-live vaccine and field strains based on nucleotide differences in thegene. The validation was performed for sensitivity and reproducibility by constructing recombinant plasmids. The limits of detection were 1.07 × 10copies/µL for the MS-H and 1.95 × 10copies/µL for field strains, respectively. The intra- and inter-assay results were less than 2.5% based on the reproducibility test. No cross-amplification signals from other common chicken pathogens were detected. Thus, our data indicated that this qPCR is sensitive, specific, and reproducible. In addition, 709 chicken clinical samples were used to evaluate this qPCR test. The results showed that positive signals could be detected from the chicken choanal cleft swabs and are 100% in concordance with the PCR sequencing method. To the best of our knowledge, we found for the first time that both L- and C-type field MS were present in flocks immunized against the MS-H vaccine strain during the validation process. In addition, this is the first report of a field strain of C-type in China.IMPORTANCE(MS) is an important pathogen in the poultry industry and has caused significant economic losses. Worldwide, an increasing number of farms are using the live attenuated vaccine MS-H strain to prevent MS infections. In order to monitor vaccinated and naturally infected flocks and to continue the MS control and eradication program, a differentiation of infected from vaccinated animals (DIVA) test for MS is urgently needed. We developed a TaqMan-MGB real-time qPCR (qPCR) method with a pair of primers and two competitive TaqMan-MGB probes. We performed an evaluation that can discriminate between the MS-H-live vaccine and field MS strains based on nucleotide differences in thegene. It has great sensitivity and reproducibility, and greater specificity than other methods which were established by SNP sites of thegene andgene.