乳牛布鲁氏菌病PCR诊断试剂盒的实验室评估

对前期研制的布鲁氏菌omp25基因-PCR试剂盒的保存期、特异性和可重复性进行了测定,并应用该试剂盒对采自宁夏、甘肃、内蒙古、山西、黑龙江、新疆省(区)的98头血清凝集试验(SAT)阳性乳牛的6批98份乳样和350头SAT阴性乳牛的2批350份乳样进行了检测。检测结果显示,PCR试剂盒与SAT的阳性符合率为100%(98/98),阴性符合率为97.71%(342/350);从SAT阴性乳牛的乳样中,用PCR试剂盒检出8份阳性,表明其敏感性高于SAT;对2株田间野毒Br-gs和Br-nmg株进行了克隆与测序,二者与标准菌株序列的同源性分别为99.8%和100%。试验结果证实,本试剂盒的可重复性良好,特异性较高,-20℃下的有效保存期为5个月。

Laboratory evaluation on PCR diagnostic kit for detecting brucellosis in dairy cow

The PCR kit for detecting omp 25 gene of Brucella abortus in raw milk was tested for its storage life, specificity and stability. Using the PCR kit, the milk samples from dairy cows in Ningxia, Gansu, Nei Monggol, Shanxi, Heilongjiang and Xinjiang provinces, China, out of which the 98 milk spe- cimens were sampled from 98 SAT-positive dairy cows, separately, and the 350 milk specimens from SAT-negative dairy cows, separately, were detected. The results showed that the positive coincident rate of the PCR kit detecting result with SAT was 100%(98/98) and the negative coincident rate of the PCR kit detecting result with SAT was 97.71%(342/350) , but the 8 sample showing negative by SAT were proved Brucella-positive by the kit, the sensitivity of the kit being higher than that of the SAT. The two field strains of Brucella omp25 genes were cloned and sequenced, sharing 99.8% and 100% nucleotide homology with that of the CCVV 12 strains, respectively, which revealed that the specificity of the kit is very reliable.