title: A rapid cycleave PCR method for distinguishing the vaccine strain Brucella abortus A19 in China.
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 polymorphism-based 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.
authors.affiliation: Laboratory of Diagnostics Development, China Animal Health and Epidemiology Center, Qingdao, Shandong Province, China (Nan, Zhang, Tan, Xu, Yiping Chen)Qingdao Agricultural University, Qingdao, Shandong Province, China (Zhang)Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu Province, China (Yuqi Chen)China Institute of Veterinary Drug Control, Beijing, China (Mao).
authors.forename: Wenlong
authors.initials: W
authors.lastname: Nan
authors.affiliation: Laboratory of Diagnostics Development, China Animal Health and Epidemiology Center, Qingdao, Shandong Province, China (Nan, Zhang, Tan, Xu, Yiping Chen)Qingdao Agricultural University, Qingdao, Shandong Province, China (Zhang)Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu Province, China (Yuqi Chen)China Institute of Veterinary Drug Control, Beijing, China (Mao).
authors.forename: Yueyong
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Laboratory of Diagnostics Development, China Animal Health and Epidemiology Center, Qingdao, Shandong Province, China (Nan, Zhang, Tan, Xu, Yiping Chen)Qingdao Agricultural University, Qingdao, Shandong Province, China (Zhang)Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu Province, China (Yuqi Chen)China Institute of Veterinary Drug Control, Beijing, China (Mao).
authors.forename: Pengfei
authors.initials: P
authors.lastname: Tan
authors.affiliation: Laboratory of Diagnostics Development, China Animal Health and Epidemiology Center, Qingdao, Shandong Province, China (Nan, Zhang, Tan, Xu, Yiping Chen)Qingdao Agricultural University, Qingdao, Shandong Province, China (Zhang)Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu Province, China (Yuqi Chen)China Institute of Veterinary Drug Control, Beijing, China (Mao).
authors.forename: Zouliang
authors.initials: Z
authors.lastname: Xu
authors.affiliation: Laboratory of Diagnostics Development, China Animal Health and Epidemiology Center, Qingdao, Shandong Province, China (Nan, Zhang, Tan, Xu, Yiping Chen)Qingdao Agricultural University, Qingdao, Shandong Province, China (Zhang)Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu Province, China (Yuqi Chen)China Institute of Veterinary Drug Control, Beijing, China (Mao).
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Laboratory of Diagnostics Development, China Animal Health and Epidemiology Center, Qingdao, Shandong Province, China (Nan, Zhang, Tan, Xu, Yiping Chen)Qingdao Agricultural University, Qingdao, Shandong Province, China (Zhang)Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu Province, China (Yuqi Chen)China Institute of Veterinary Drug Control, Beijing, China (Mao).
authors.forename: Kairong
authors.initials: K
authors.lastname: Mao
authors.affiliation: Laboratory of Diagnostics Development, China Animal Health and Epidemiology Center, Qingdao, Shandong Province, China (Nan, Zhang, Tan, Xu, Yiping Chen)Qingdao Agricultural University, Qingdao, Shandong Province, China (Zhang)Xi'an Jiaotong-Liverpool University, Suzhou, Jiangsu Province, China (Yuqi Chen)China Institute of Veterinary Drug Control, Beijing, China (Mao) cyp777@163.com.
authors.forename: Yiping
authors.initials: Y
authors.lastname: Chen
mesh_terms: D000818:Animals; D002004:Brucella Vaccine / Q000008:administration & dosage*; D002003:Brucella abortus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D002006:Brucellosis / Q000097:blood / Q000382:microbiology / Q000517:prevention & control / Q000662:veterinary*; D002417:Cattle; D002681:China; D006041:Goats; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary; D011237:Predictive Value of Tests; D013552:Swine; D014611:Vaccination / Q000662:veterinary
chemical_list: D002004:Brucella Vaccine
keywords: B. abortus A19; B. abortus S19; Brucella; cycleave PCR