马梨形虫病双重PCR检测方法的建立
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摘要
为了建立马梨形虫病双重PCR检测方法,试验根据Gen Bank发表的驽巴贝斯虫和马泰勒虫18S rRNA保守基因序列分别合成2对特异性引物,以核酸混合物为模板,优化反应条件建立马梨形虫病双重PCR检测方法,并检验该方法的特异性和敏感性。同时用该双重PCR检测方法对采自昭苏种马场的马疑似病例全血进行检测,并与镜检法和单重PCR法进行比较。结果表明:该双重PCR检测方法能对驽巴贝斯虫和马泰勒虫核酸扩增出大小为529 bp和789 bp特异性目的片段,而对双芽巴贝斯虫、环形泰勒虫、羊泰勒虫、牛巴贝斯虫核酸的扩增均为阴性;驽巴贝斯虫和马泰勒虫阳性DNA被稀释1×108倍时均能检出其相应目的片段;对采集的46份马疑似病例血样进行PCR诊断,其中驽巴贝斯虫感染率为30.4%(14/46),马泰勒虫感染率为41.3%(19/46);双重PCR检测方法与单重PCR方法的符合率为100%。说明建立的双重PCR检测方法具有一定的特异性和敏感性,可用于驽巴贝斯虫和马泰勒虫临床感染隐性病例的联合检测与鉴别诊断。
The aim of the present study was toestablish a duplex PCR method for detection of equine piroplasma.According to the 18 S rRNA conserved gene sequences of Babesia caballi and Theileria equi published by Gen Bank,two pairs of specific primers were synthesized. Then,using nucleic acid mixture as template,the reaction conditions were optimized to establish a duplex PCR assay method for detection of equine piroplasma,and the specificity and sensitivity of the method were tested.At the same time,the horse blood samples of suspected cases collected from Zhaosu equestrian farm were detected by the duplex PCR method,and compared with the microscopic examination and routine PCR method.The results showed that the specific fragments of 529 bp and 789 bp were amplified by using the duplex PCR from DNA samples of B.caballi and T.equi,respectively. But the nucleic acid amplification results of B.bigemina,T.annulata,T. sergenti and B. bovis were negative. When the positive DNA concentrations of both B.caballi and T. equi were diluted 1 × 10~8 fold,the corresponding target fragments could still be detected. 46 blood samples collected from suspected cases of horses were diagnosed by PCR. The infection rate of B.caballi was 30. 4%( 14/46). The infection rate of T.equi was 41. 3%( 19/46). The coincidence rate between the results of duplex PCR and the routine PCR was 100%.The results indicate that the duplex PCR detection method has certain specificity and sensitivity,and can be used for the detection and differential diagnosis of recessive cases of clinical infection of B.caballi and T.equi.
The aim of the present study was toestablish a duplex PCR method for detection of equine piroplasma.According to the 18 S rRNA conserved gene sequences of Babesia caballi and Theileria equi published by Gen Bank,two pairs of specific primers were synthesized. Then,using nucleic acid mixture as template,the reaction conditions were optimized to establish a duplex PCR assay method for detection of equine piroplasma,and the specificity and sensitivity of the method were tested.At the same time,the horse blood samples of suspected cases collected from Zhaosu equestrian farm were detected by the duplex PCR method,and compared with the microscopic examination and routine PCR method.The results showed that the specific fragments of 529 bp and 789 bp were amplified by using the duplex PCR from DNA samples of B.caballi and T.equi,respectively. But the nucleic acid amplification results of B.bigemina,T.annulata,T. sergenti and B. bovis were negative. When the positive DNA concentrations of both B.caballi and T. equi were diluted 1 × 10~8 fold,the corresponding target fragments could still be detected. 46 blood samples collected from suspected cases of horses were diagnosed by PCR. The infection rate of B.caballi was 30. 4%( 14/46). The infection rate of T.equi was 41. 3%( 19/46). The coincidence rate between the results of duplex PCR and the routine PCR was 100%.The results indicate that the duplex PCR detection method has certain specificity and sensitivity,and can be used for the detection and differential diagnosis of recessive cases of clinical infection of B.caballi and T.equi.
引文
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[11]张松林,刘磊,张太翔,等.欧洲型和美洲型PRRSV双重PCR监测方法的建立[J].动物医学进展,2012,33(12):26-30.
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[2]QABLAN M A,SLOBODA M,JIRKU M.Quest for the piroplasms in camels:identification of Theileria equi and Babesia caballi in Jordanian dromedaries by PCR[J].Vet Parasitol,2012,186(3/4):456-460.
[3]RIBEIRO I B,CAMARA A C,BITTENCOURT M V,et al.Detection of Theileria equi in spleen and blood of asymptomatic piroplasm carrier horses[J].Acta Parasitol,2013,58(2):218-222.
[4]MACHADO R Z,TOLEDO C Z,TEIXEIRA M C,et al.Molecular and serological detection of Theileria equi and Babesia caballi in donkeys(equus asinus)in Brazil[J].Vet Parasitol,2012,186(3/4):461-465.
[5]李佳.马泰勒虫荧光定量PCR检测方法的建立及伊犁马泰勒虫病的分子流行病学调查[D].乌鲁木齐:新疆农业大学,2013.
[6]罗金,刘光远,谢俊仁,等.驽巴贝斯虫病PCR检测方法的建立和应用[J].中国畜牧兽医,2012,38(7):193-195.
[7]肖雯.绵羊痘病毒和山羊痘病毒双重PCR检测方法的建立[D].重庆:西南大学,2012.
[8]刘启生,王振宝,王真,等.牛巴贝斯虫实时荧光PCR检测方法的建立[J].中国预防兽医学报,2013,35(1):58-61.
[9]王振宝,刘启生,哈森,等.牛环形泰勒虫病二温式PCR诊断方法的建立及初步应用[J].动物医学进展,2012,33(5):59-63.
[10]张杨,达伊力特,刘进,等.驽巴贝斯虫二温式PCR检测方法的建立及初步应用[J].动物医学进展,2014,35(6):16-20.
[11]张松林,刘磊,张太翔,等.欧洲型和美洲型PRRSV双重PCR监测方法的建立[J].动物医学进展,2012,33(12):26-30.
[12]向智龙,陈振涛,卓建华,等.山羊痘病毒和羊口疮病毒双重PCR监测方法的建立与初步应用[J].中国畜牧兽医,2011,38(1):88-91.