摘要
用２对已发表的引物和１对自行设计的引物对同一禽传染性支气管炎病毒（ＩＢＶ）Ｈ１２０株进行ＲＴ－ＰＣＲ，分别获得了Ｓ１基因上与引物设计相一致的１７２０、２２８、６０２ｂｐ的扩增片段。用自行设计的引物对７个毒株（Ｈ１２０、Ｈ５２、Ｍ４１、Ｃｏｎｎ、Ｇｒａｙ、Ｔ、Ｈｏｌｔｅ）和５个分离株（宜毒、上毒、云毒、ＨＫ、１１８）的含毒尿囊液或纯化病毒进行ＲＴ－ＰＣＲ，结果除Ｈｏｌｔｅ株和２个分离株（宜毒、云毒）外，其余均成功地扩增出６００ｂｐ的片段。用１．７、０．２、０．６ｋｂ３对引物对６个ＩＢＶ毒株和６个分离株的含毒尿囊液在相同和不同条件下进行ＲＴ－ＰＣＲ，结果３对引物分别扩增出３、５、９株ＩＢＶ，同时可将不同血清型的１２个ＩＢＶ株分成６种基因型。将ＩＢＶ分离株ＨＫ与标准株Ｍ４１经ＰＣＲ扩增、ＨａｅⅢ和Ｈｉｎｄ酶切、ＲＦＬＰ分析，表明属同一马萨诸塞血清型。３株ＩＢＶ（Ｈ１２０，ＨＫ，Ｍ４１）在鸡胚中繁殖，ＰＣＲ最早检出的时间为２０～２４ｈ。ＲＴ－ＰＣＲ提供了直接从尿囊液和感染鸡组织中快速检测病毒的新方法。
Reverse transcription(RT) of viral RNA and polymerase chain reaction (PCR) were
used to amplify the IBV H120 viral genome using two pairs of primers previously published and
a pair of selfdesigned primers. Three special PCR fragments of 1 720 bp, 228 bp and 602 bp
corresponding to the three primers were obtained respectively. The PCR products which
contained the S1 glycoprotein gene of IBV were of the expected size. The IBV viral RNA
extracted from the allantoic fluid of infected embronating eggs or purified virus from 7 different
IBV reference strains and 5 field strains were amplified by RTPCR using the selfdesigned
primer. Except one reference strain and 2 field strains, all the other strains were successfully
amplified. The PCR products appeared to be approximate 600 bp, the expected size. The
allantoic fluid of infected embryonating eggs of 6 IBV reference strains and 6 field strains were
amplified by RTPCR using three pairs of primers of 1.7, 0.2, 0.6 kb on same and different
condition, 3, 5, 9 strains of IBV were amplified respectively. While 12 different sentypes of the
12 IBV strains can be divided in 6 genotypes. The PCR amplification products of IBV isolated
strain HK and standard strain M41 were digested with restriction enzymes Hae and Hind . The
results showed that the two IBV strain were grouped the same Massachuset serotype according
to the RFLP patterns. The RTPCR technique provided a new method to directly detect the IBV
from the allantoic fluid of infected embryonating eggs and tissues of infected chickens.
出处
《中国兽医学报》
CAS
CSCD
北大核心
1999年第3期237-240,共4页
Chinese Journal of Veterinary Science
