高敏荧光层析法在骆驼布鲁氏菌病流行病学调查中的应用

Application of a high-sensitivity lanthanide fluorescence chromatography method in epidemiological investigation of brucellosis in camels

目的了解新疆部分地区骆驼布鲁氏菌病(简称布病)流行情况,探索高敏荧光层析法在骆驼布病流行病学调查中的应用。方法分别在新疆阿勒泰地区、塔城地区、和田地区、伊犁地区、博尔塔拉蒙古自治州采集骆驼血清2000份,骆驼乳307份。应用布病虎红平板凝集试验(RBT)和试管凝集试验(SAT)进行骆驼布病血清学调查。提取骆驼乳基因组DNA,应用实时荧光PCR(Realtime-PCR)和AMOS-PCR方法进行病原学检测并分型。同时应用高敏荧光层析法对200份骆驼血清和100份骆驼乳样进行布病符合率检测。结果新疆5个地区骆驼血清SAT确诊阳性31份,布病血清学阳性率为1.55%(31/2000)。307份骆驼乳经Realtime-PCR检测,5份骆驼乳出现扩增曲线且Ct值在29.65~33.68,骆驼乳布鲁氏菌核酸阳性率为1.63%(5/307)。其中2份骆驼乳提取基因组DNA经AMOS-PCR扩增获得498 bp目的条带,测序并在NCBI进行BLAST比对分析,结果与牛种生物I型布鲁氏菌序列同源性在99.8%~100%。高敏荧光层析法检出骆驼布病阳性血清33份,阴性血清167份,该方法敏感性为96.77%,特异性为98.22%,与SAT检测方法总体符合率为98%,两种方法的Kappa值为0.925。高敏荧光层析法对100份骆驼乳检出布病抗体阳性乳7份,阴性乳93份,该方法与病原学检测方法(Realtime-PCR)总体符合率为98%。结论布鲁氏菌高敏荧光层析法具有灵敏度和特异度高的优点,且与布病常规检测方法符合率较好,可用于骆驼布病现场快速检测。骆驼布病流行病学调查数据提示应加强骆驼布病防控,同时加强骆驼乳的食品安全监管。

This study was aimed at determining the prevalence of brucellosis in camels in some areas of Xinjiang and to establish an epidemiological investigation method for camel Brucellosis.A total of 2000 camel serum and 307 camel milk samples were collected from the Altai,Tacheng,Hotan,Yili and Bortala Mongolian Autonomous Regions in Xinjiang.Serological investigation of brucellosis in camels was performed with Rose Bengal test(RBT)and serum agglutination test(SAT).The genomic DNA extracted from camel milk was detected and typed by real time-PCR and AMOS-PCR.High-sensitivity lanthanide fluorescence chromatography was performed on 200 camel serum and 100 camel milk samples for Brucellosis compliance testing.A total of 31 camel sera were confirmed positive for SAT in five regions of Xinjiang,thus resulting in a seropositivity rate of 1.55%(31/2000).A total of 307 camel milk samples were tested by real time-PCR,five of which showed an amplification curve with Ct values from 29.6 to 33.6,thus resulting in a positivity rate of 1.63%(5/307)for nucleic acid testing in camel milk.Two camel milk samples showed 498 bp target bands after genomic DNA amplification by AMOS-PCR;these bands were sequenced and analyzed with BLAST in NCBI and showed 99.8%-100%sequence identity with Brucella bovis type I.33 positive serum and 167 negative serum samples were subjected to high-sensitivity lanthanide fluorescence chromatography for brucellosis in camels;the sensitivity was 96.77%,and the specificity was 98.81%.The overall coincidence rate with SAT was 98%.The kappa value for the two methods was 0.925.High-sensitivity lanthanide fluorescence chromatography was applied to camel milk,and 7 brucellosis antibody positive and 93 brucellosis antibody negative samples were detected out of 100 camel milk samples.The overall coincidence rate for this method and the pathogen detection method(real time-PCR)was 98%.This high-sensitivity fluorescence chromatography method has advantages of high sensitivity and specificity,as well as better compliance with the routine detection method of Brucella.This method is suitable for rapid field detection of brucellosis in camels.The data from an epidemiological survey of brucellosis in camels suggest that prevention and control of brucellosis in camels,as well as food safety control of camel milk,should be strengthened.