Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B-canis DNA in canine blood samples
Development and evaluation of a seminested PCR for detection and differentiation of Babesia gibsoni (Asian genotype) and B-canis DNA in canine blood samples
复制标题
DOI：
10.1128/jcm.41.9.4172-4177.2003
复制
发表时间：
2003-09-01
影响因子：
9.4
通讯作者：
Breitschwerdt, EB
中科院分区：
文献类型：
--
作者：
Birkenheuer, AJ;Levy, MG;Breitschwerdt, EB
犬巴贝斯虫病最近被认为是一种新兴的传染病的狗在北美。我们试图建立一种检测和区分吉氏巴氏巴氏杆菌（亚洲基因型）B的双套式PCR。犬亚种vogeli，B.犬亚种犬和B.犬亚种在狗的血液样本中发现了罗西基因设计一对外引物，从B中扩增出与18 S rRNA基因相似的340 bp片段。gibsoni（亚洲基因型）、B.犬亚种沃盖蒂湾，B.犬亚种rossi和B.犬亚种而不是哺乳动物的DNA。设计正向引物，其将在套式PCR中从每种生物体特异性扩增较小片段。实际检测限为50个微生物/ml模拟感染EDTA抗凝全血。引物对也扩增出了一个类似于B的370 bp片段。gibsoni（USA/加州基因型）18 S rRNA基因，其来自具有高百分比寄生虫血症的实验感染的狗的血液。扩增子未检测到当DNA提取的血液中的狗，自然感染了泰勒虫在低百分比的寄生虫血症进行了扩增。由于灵敏度有限，不建议将该试验用于B的常规诊断。gibsoni（USA/加州基因型）或T.安东在任何测试条件下，PCR检测均未扩增弓形虫、犬新孢子虫、婴儿利什曼原虫、微小隐孢子虫或犬DNA。双套式PCR试验能够检测和区分B。gibsoni（亚洲基因型）、B.犬亚种vogeli，B.犬亚种犬和B.犬亚种从受感染的狗的血液样本中检测到罗西DNA。
Canine babesiosis has recently been recognized as an emerging infectious disease of dogs in North America. We sought to develop a seminested PCR to detect and differentiate Babesia gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in canine blood samples. An outer primer pair was designed to amplify an similar to340-bp fragment of the 18S rRNA genes from B. gibsoni (Asian genotype), B. canis subsp. vogeti, B. canis subsp. rossi, and B. canis subsp. canis but not mammalian DNA. Forward primers were designed that would specifically amplify a smaller fragment from each organism in a seminested PCR. The practical limit of detection was 50 organisms/ml of mock-infected EDTA anticoagulated whole blood. The primer pair also amplified an similar to370-bp fragment of the B. gibsoni (USA/California genotype) 18S rRNA gene from the blood of an experimentally infected dog with a high percentage of parasitemia. Amplicons were not detected when DNA extracted from the blood of a dog that was naturally infected with Theileria annae at a low percentage of parasitemia was amplified. Due to limited sensitivity, this test is not recommended for the routine diagnosis of B. gibsoni (USA/California genotype) or T. annae. The PCR test did not amplify Toxoplasma gondii, Neospora caninum, Leishmania infantum, Cryptosporidium parvum, or canine DNA under any of the conditions tested. The seminested PCR test was able to detect and discriminate B. gibsoni (Asian genotype), B. canis subsp. vogeli, B. canis subsp. canis, and B. canis subsp. rossi DNA in blood samples from infected dogs.