2006 年至 2007 年巴基斯坦旁遮普省口蹄疫血清型 O 的 RT-PCR 鉴定和序列分析。
RT-PCR evaluation for identification and sequence analysis of foot-and-mouth disease serotype O from 2006 to 2007 in Punjab, Pakistan.
机构信息
National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
出版信息
Comp Immunol Microbiol Infect Dis. 2011 Mar;34(2):95-101. doi: 10.1016/j.cimid.2009.10.004. Epub 2009 Dec 23.
FMD clinically positive 250 tissue samples (mouth and hoof epithelium and vesicle swabs, tongue tissue) and 175 secretion samples (milk, saliva, serum, plasma) were evaluated by RT-PCR for the diagnosis of FMD with different pair of universal and serotype-specific primers from 2006 to 2007 in Punjab, Pakistan. Universal primer pair P1/P2 from VP1 gene detected FMD in 182 out of 250 (72.8%) tissues and 92 out of 175 (52.6%) secretion samples, while universal primer 1F/1R from 5'UTR region detected FMD in 218 out of 250 (87.2%) tissues and 142 out of 175 (81.1%) secretion samples. 1F/1R proved better than the P1/P2 primer pair for primary diagnosis of FMD, direct from the clinical positive samples. Direct sequencing of the universal primer pair P1/P2 revealed that O serotype of FMD was circulating in this region. O serotype of FMD was detected with O-1C(ARS4)/PNK 61, AU(O)/AU(rev), AU(O)/PNK61 primer pairs, these primer pairs also compared with each other. AU(O)/AU(rev) and AU(O)/PNK61 detected O serotype of FMD in 88.9% tissue and swab (mouth and hoof vesicle swabs) samples and 71.9% different secretion (milk, saliva, serum, plasma) samples, while O-1C(ARS4)/PNK 61 detected 48.1% tissue and swab (mouth and hoof vesicle swabs) samples and 37.5% different secretion (milk, saliva, serum, plasma) samples. AU(O)/AU(rev), AU(O)/PNK61 primer pairs detected 40.8% more tissue and swab samples, while these pairs detected 34.4% more secretion samples. Cloning of PCR product of AU(O)/AU(rev) VP1 gene and sequencing for phylogenetic studies revealed that O serotype of FMD circulating in Punjab, Pakistan was genetically very diverse from the 'O' serotype in Middle East and Europe. The dendrogram showed that Pakistan 'O' serotype was very much similar genetically to its neighbor countries (Sri Lanka, India, Iran, Iraq, and China) and PanAsia 1 lineage which caused 2001-outbreak in UK and 1994-outbreak in Saudi Arabia, etc.
2006 年至 2007 年，在巴基斯坦旁遮普省，使用来自 VP1 基因的通用引物对 P1/P2 和 5'UTR 区的通用引物 1F/1R，对来自 250 份（口腔和蹄上皮和水疱拭子、舌组织）临床阳性组织样本和 175 份（牛奶、唾液、血清、血浆）分泌样本进行了不同的通用和血清型特异性引物的 RT-PCR 检测，以诊断口蹄疫。通用引物对 P1/P2 从 250 份组织样本中检测到口蹄疫 182 份（72.8%），从 175 份分泌样本中检测到口蹄疫 92 份（52.6%），而通用引物 1F/1R 从 250 份组织样本中检测到口蹄疫 218 份（87.2%），从 175 份分泌样本中检测到口蹄疫 142 份（81.1%）。1F/1R 比 P1/P2 引物对更适合直接从临床阳性样本中对口蹄疫进行初步诊断。对通用引物对 P1/P2 的直接测序显示，该地区有 O 型口蹄疫在传播。使用 O-1C(ARS4)/PNK 61、AU(O)/AU(rev)、AU(O)/PNK61 引物对检测到 O 型口蹄疫，还比较了这些引物对之间的差异。AU(O)/AU(rev) 和 AU(O)/PNK61 在 88.9%的组织和拭子（口腔和蹄部水疱拭子）样本中检测到 O 型口蹄疫，在 71.9%的不同分泌（牛奶、唾液、血清、血浆）样本中检测到 O 型口蹄疫，而 O-1C(ARS4)/PNK 61 在 48.1%的组织和拭子（口腔和蹄部水疱拭子）样本中检测到 O 型口蹄疫，在 37.5%的不同分泌（牛奶、唾液、血清、血浆）样本中检测到 O 型口蹄疫。AU(O)/AU(rev)、AU(O)/PNK61 引物对检测到的组织和拭子样本分别增加了 40.8%和 34.4%，而这些引物对检测到的分泌样本分别增加了 34.4%。对 AU(O)/AU(rev) VP1 基因的 PCR 产物进行克隆和系统发育研究，结果表明，在巴基斯坦旁遮普省流行的 O 型口蹄疫与中东和欧洲的“O”型口蹄疫在遗传上有很大的不同。系统发育树显示，巴基斯坦的“O”型口蹄疫在遗传上与邻国（斯里兰卡、印度、伊朗、伊拉克和中国）和泛亚 1 谱系非常相似，该谱系导致了 2001 年英国爆发和 1994 年沙特阿拉伯爆发等疫情。
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