A rapid cycleave PCR method for distinguishing the vaccine strain Brucella abortus A19 in China

Abstract

Abstract. Brucellosis is a widespread zoonotic disease caused by Brucella spp. Immunization with attenuated vaccines has proved to be an effective method of prevention; however, it may also interfere with diagnosis. Brucella abortus strain A19, which is homologous to B. abortus strain S19, is widely used for the prevention of bovine brucellosis in China. For effective monitoring of the control of brucellosis, it is essential to distinguish A19 from field strains. Single-nucleotide polymorphismbased assays offer a new approach to such discrimination studies. In the current study, we developed a cycleave PCR assay that successfully distinguished attenuated vaccine strains A19 and S19 from 22 strains of B. abortus and 57 strains of 5 other Brucella species. The assay gave a negative reaction with 4 non-Brucella species. The minimum sensitivity of the assay, evaluated using 10-fold dilutions of chromosomal DNA, was 7.6 fg for the A19 strain and 220 fg for the single non-A19/non-S19 Brucella strain tested (B. abortus 104M). The assay was also reproducible (intra-and interassay coefficients of variation: 0.003-0.01 and 0.004-0.025, respectively). The cycleave assay gave an A19/S19-specific reaction in 3 out of 125 field serum samples, with the same 3 samples being positive in an alternative A19/S19-specific molecular assay. The cycleave assay gave a total of 102 Brucella-specific reactions (3 being the A19/S19-specific reactions), whereas an alternative Brucella-specific assay gave 92 positive reactions (all also positive in the cycleave assay). Therefore, this assay represents a simple, rapid, sensitive, and specific tool for use in brucellosis control.

Combined nucleic acid assays for diagnosis of A19 vaccine‐caused human brucellosis

“…There are reports of the establishment of SNP‐based MGB methods that can be used to quickly identify A19, RB51 and Rev.1 vaccine strains (Gopaul et al., 2010). Wenlong Lan et al established a cycleave PCR, which can accurately and reliably identify A19/S19 and non‐A19/S19 and is 10 times more sensitive than the MGB method (Nan et al., 2016). However, this detection method requires expensive instruments and reagents and is difficult to use in routine clinical diagnosis (Franco, Mulder, Gilman, & Smits, 2007).…”

“…Therefore, it is of great significance to determine whether an animal or human that has been exposed to A19 is infected. Due to which can accurately and reliably identify A19/S19 and non-A19/ S19 and is 10 times more sensitive than the MGB method (Nan et al, 2016). However, this detection method requires expensive instruments and reagents and is difficult to use in routine clinical diagnosis (Franco, Mulder, Gilman, & Smits, 2007).…”

“…The most effective are attenuated live vaccines, which mainly include Brucella abortus strain A19 (the most commonly used in Chinese cattle), Brucella suis strain S2, Brucella melitensis strains M5 and Rev.1. Despite the good results, there are also some disadvantages: A19 can cause the miscarriage of pregnant females and cause minor infections in humans (Lalsiamthara & Lee, 2017; Nan et al., 2016). Therefore, it is necessary to distinguish the A19 vaccine from other wild strains in farms that have cattle immunized with A19, thus eliminating animals that have been infected with wild strains.…”

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

“…The recent development of SNP-based real-time assays offers a new approach for overcoming this hurdle. SNP-based 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) [ 12 , 13 ].…”

PCR Based Molecular Diagnostic Assays for Brucellosis: A Review

“…The present real-time PCR showed greater sensitivity than that of conventional PCR and previously described TaqMan probe based real-time PCR assays which make it a valuable tool for differentiating B. abortus infection with rapidity and accuracy. Nan et al, (2016) developed a rapid cycleave PCR assay for differentiating the vaccine strain Brucella abortus A19 from field strains. This study was designed on SNP (C 587 -T 587 ) in BAbS19_I07270 (arginyl-transfer RNAprotein transferase) locus.…”