科研動(dòng)態(tài)

鸚鵡幼雛病是由禽類多瘤病毒（APV）引起的多種鸚鵡雛鳥死亡的急性病毒性傳染病，嚴(yán)重危害鸚鵡養(yǎng)殖業(yè)的健康發(fā)展。為提高分子生物學(xué)方法檢測APV的敏感性和特異性，對APV基因片段進(jìn)行克隆和序列分析，設(shè)計(jì)合成1對特異性引物，以VP1基因?yàn)槟０澹?jīng)PCR擴(kuò)增獲得731 bp的核苷酸DNA，并用DIG標(biāo)記，制備用于檢測APV的特異性核酸探針；對制備的探針進(jìn)行靈敏度檢測，同時(shí)與普通PCR進(jìn)行敏感性比較；使用制備的探針，對經(jīng)分離鑒定和制備保存的其他7種禽病毒核酸進(jìn)行特異性檢測；用該核酸探針，對疑似感染APV的鸚鵡病料進(jìn)行斑點(diǎn)雜交檢測，并對鑒定為陽性的APV進(jìn)行全基因組擴(kuò)增和序列分析。結(jié)果顯示：該探針可檢測到2 pg量的APV特異性核酸片段；僅APV-VP1陽性核酸顯色，呈現(xiàn)陽性反應(yīng)，而陰性核酸和其他7種禽病毒核酸均不顯色，呈陰性反應(yīng)。結(jié)果表明：建立的核酸斑點(diǎn)雜交檢測方法具有較高的靈敏度和特異性，可用于臨床初步診斷。本方法的建立為我國開展APV分子流行病學(xué)調(diào)查及其感染的臨床診斷提供了技術(shù)支撐。

Establishment and Application of PCR Combined with Dot Blot Hybridization for Detecting Avian Polyomavirus

Budgerigar fledgling disease（BFD）is an acute viral infectious disease caused by avian polyomavirus（APV），which could lead to death of various parrot nestlings and seriously endanger the healthy development of parrot industry. In order to improve the sensitivity and specificity of molecular biological methods to detect APV，the APV gene fragment was cloned and sequenced. After designing and synthesizing a pair of specific primers as well as carrying out PCR amplification，gene fragment with the nucleotide length of 731 bp was obtained by taking VP1 gene as a template，and then the fragment was marked with DIG to prepare a specific probe for detecting APV；the sensitivity of the probe was detected and compared with general PCR；other 7 kinds of isolated and prepared virus nucleic acids were used to evaluate the specificity of the probe；the lesions of parrots suspected to be infected with APV were detected by dot blot hybridization using the probe，and the whole genome sequences of the positive APV were amplified and analyzed. The results showed that the probe could detect 2 pg of APV specific nucleic acid fragment；and only the positive nucleic acids of APV-VP1 showed positive reaction，while the negative one and other 7 kinds of virus nucleic acids all showed negative reaction. In conclusion，the established method could be used for clinical preliminary diagnosis due to its high sensitivity and specificity. Therefore，molecular epidemiological investigation and clinical diagnosis of APV infection in China were provided with technical supports by the established method.  

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