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BackgroundBrucellosis is a widespread zoonotic disease caused by Gram-negative Brucella bacteria. Immunisation with attenuated vaccine is an effective method of prevention, but it can interfere with diagnosis. Live, attenuated Brucella abortus strain 104M has been used for the prevention of human brucellosis in China since 1965. However, at present, no fast and reliable method exists that can distinguish this strain from field strains. Single nucleotide polymorphism (SNP)-based assays offer a new approach for such discrimination. SNP-based minor groove binder (MGB) and Cycleave assays have been used for rapid identification of four Brucella vaccine strains (B. abortus strains S19, A19 and RB51, and B. melitensis Rev1). The main objective of this study was to develop a PCR assay for rapid and specific detection of strain 104M.ResultsWe developed a SNP-based MGB PCR assay that could successfully distinguish strain 104M from 18 representative strains of Brucella (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae, and B. ovis), four Brucella vaccine strains (A19, S19, S2, M5), and 55 Brucella clinical field strains. The assay gave a negative reaction with four non-Brucella species (Escherichia coli, Pasteurella multocida, Streptococcus suis and Pseudomonas aeruginosa). The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 220 fg for the 104M strain and 76 fg for the single non-104M Brucella strain tested (B. abortus A19). The assay was also reproducible (intra- and inter-assay coefficients of variation = 0.006–0.022 and 0.012–0.044, respectively).ConclusionsA SNP-based MGB PCR assay was developed that could straightforwardly and unambiguously distinguish B. abortus vaccine strain 104M from non-104M Brucella strains. Compared to the classical isolation and identification approaches of bacteriology, this real-time PCR assay has substantial advantages in terms of simplicity and speed, and also reduces potential exposure to live Brucella. The assay developed is therefore a simple, rapid, sensitive, and specific tool for brucellosis diagnosis and control.

Feline calicivirus (FCV) is a highly contagious pathogen that can cause seriously upper respiratory tract and oral disease in feline. Despite widespread vaccination, the prevalence of FCV remains high. In this study, the FCV qd/2019/china was isolated from domestic feline oropharyngeal swab which was collected in qingdao, China. The virus was purified with plaque assay and identified with PCR and IFA method. the capsid amino acid ,VP1, of qd/2019/china [1] showed sequence identity with other isolate ranging from 83.9% (ym3/2001/jp) to 91.1% (CH-JL4) .Sequence analysis of the capsid amino acid revealed that qd/2019/china was closely related to CH-JL4 and clustered with CH-JL4 in the phylogenetic tree. Phylogenetic analysis indicated that complete genome of qd/2019/china and CH-JL4 was also classified into the same cluster. While the recombination analysis with Simplot indicate that the qd/2019/china originated from the recombination of CH-JL4 and HRB-SS, and the region 3821–5301 nt originated from HRB-SS. Further, the region 3821–5301 nt belong to protease -polymerase (PP) of HRB-SS. Here we isolate a new FCV qd/2019/china, which may be a recombinant virus, These results were beneficial for understanding the evolution of FCV.