Reverse transcription-polymerase chain reaction on pooled samples to detect bovine viral diarrhea virus by using fresh ear-notch-sample supernatants
Reverse transcription-polymerase chain reaction on pooled samples to detect bovine viral diarrhea virus by using fresh ear-notch-sample supernatants
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DOI：
10.1177/104063870601800113
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发表时间：
2006-01-01
影响因子：
1.5
通讯作者：
Powers, B
中科院分区：
文献类型：
--
作者：
Kennedy, JA;Mortimer, RG;Powers, B
从3，599头一岁小母牛中采集耳切口样本，以检测疑似牛病毒性腹泻病毒（BVDV）持续感染（PI）的动物。个体免疫组织化学（IHC）、个体抗原捕获酶联免疫吸附试验（AC-ELISA）和逆转录-聚合酶链反应（RT-PCR）检测合并耳切口上清液与来自3，016头小母牛的样本进行了比较，而RT-PCR耳切口池和个体AC-ELISA检测与来自所有3，599头小母牛的样本进行了比较。通过IHC和AC-ELISA鉴定出4头小母牛为阳性。而其余的小母牛通过两种测试均被鉴定为阴性。当随机合并来自100只动物的耳切口的上清液并对每个池完成RT-PCR时，RT-PCR鉴定出含有1个阳性AC-ELISA样品的2个池和含有2个阳性AC-ELISA样品的1个池。对合并的RT-PCR耳切口上清液的进一步评价检测到100%（n = 36）加标来自单个随机选择的阳性AC-ELISA耳切口的上清液的样品。尽管未完成后续确证性试验，但所有3种方法在检测可疑PI动物方面均100%相关，kappa值为1。对合并的耳切口上清液使用RT-PCR可提供一种初步、快速、经济有效的方法，用于筛选牛群中的BVDV PI动物。随后采用AC-ELISA进行系列检测，以评估阳性样本池中的单个样本，可最大限度地缩短其他动物暴露于病毒的时间。
Ear-notch samples from 3,599 yearling heifers were collected to detect persistently infected (PI) animals with suspect bovine viral diarrhea virus (BVDV). Individual immunohistochemistry (IHC), individual antigen-capture enzyme-linked immunosorbent assay (AC-ELISA), and reverse transcription-polymerase chain reaction (RT-PCR) tests with pooled ear-notch supernatants were compared with samples from 3,016 heifers, whereas RT-PCR ear-notch pools and individual AC-ELISA tests were compared with samples from all 3,599 heifers. Four heifers were identified positive by both IHC and AC-ELISA. whereas the remaining heifers were identified negative by both tests. When supernatant from ear notches from 100 animals was randomly pooled and RT-PCR was accomplished on each pool, RT-PCR identified 2 pools that contained I positive AC-ELISA sample and I pool that contained 2 positive AC-ELISA samples. Further evaluation of the pooled RT-PCR ear-notch supernatant detected 100% (n = 36) samples spiked with supernatant from a single randomly selected positive AC-ELISA ear notch. Although follow-up confirmatory tests were not completed, all 3 methods correlated 100% in detecting suspect PI animals, with a kappa value of 1. The use of RT-PCR on pooled ear-notch supernatant could provide an initial, rapid, cost-effective method of screening cattle herds for BVDV PI animals. Subsequent serial testing with an AC-ELISA to evaluate individual samples included in the positive pool could minimize the length of time other animals are exposed to the virus.