A rapid minor groove binder PCR method for distinguishing the vaccine strain Brucella abortus 104M

Abstract

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.

Diagnóstico de Brucelosis en caprinocultores y su ganado caprino en ejidos de la Comarca Lagunera

“…La Brucelosis es una enfermedad infectocontagiosa, aguda o crónica, que afecta tanto a los animales como al hombre, causada por bacterias del género Brucella (Nan et al 2018). Conocida antiguamente como fiebre de Malta, fiebre del Mediterráneo, fiebre ondulante, enfermedad de Bang, y más recientemente nombrada por el organismo que la produce, la brucelosis es considerada una de las zoonosis más relevantes en el concierto mundial hoy en día (Guzmán-Hernández et al 2016).…”

“…B. melitensis afecta fundamentalmente a cabras y ovejas, pero puede afectar a bóvidos y cerdos. Es una zoonosis que afecta a rumiantes, camellos, cerdos, perros y diversas formas de vida terrestre y marina, causando aborto, infertilidad y su subsecuente pérdida económica en la ganadería (Fiorentino, Paolicchi et al 2018;Nan, Qin et al 2018;Salvador-Bescós, Gil-Ramírez et al 2018). Es la responsable de la gran mayoría de los casos en el hombre, ocasionando además los de mayor gravedad.…”