A duplex polymerase chain reaction assay(dRT-PCR)was developed in this study which could simultaneously ampli- fy the hemagglutinin glycoprotein (HA) gene of the AIV H5 and distinctive sequence of vNDV F gene in a single tube. The positive results could be obtained from AIV H5 and vNDV strains while no positive amplification was observed with other HA subtypes of AIV, avirulent NDV and other unrelated avian pathogens. The endpoint of detection was defined as approxi- mately 20.6fg for DNA plasmid containing HA gene of AIV H5 and 406fg for DNA plasmid containing F gene of vNDV. Compared with the single RT-PCR, the dRT-PCR assay was able to detect AIV/NDV with similar sensitivity. The whole process of detection, from sample processing to obtaining the results, could be finished within 5h. Of 24 clinical samples detected by this assay, all were AIV-H5 negative and 18 were vNDV positive. Sequencing analysis confirmed that all PCR products contained characteristic sequence of vNDV at cleavage site. NDV isolation was performed by SPF embryo from 9 clinical sam- ples randomly selected and showed a 6/9 isolation rate. Samples of livers, brains, lungs and cloacal swabs collected from 5 five-day-old SPF chicken experimentally co-infected with 100ELD50 AIV H5 and 100ELD50 vNDV were detected by the dRT-PCR, the detection rate of AIV H5 was 4/5,3/5,5/5, 4/5, the detection rate of vNDV was 3/5,5/5,4/5,3/5; while no positive samples from chicken experimentally co-infected with AIV H9N2 and LaSota, and negative control has been found using the dRT-PCR, In conclusion, the method developed in this study provides a rapid, accurate, economical and effective way to detect AIV H5 and vNDV.

According to hemagglutinin (HA) gene of H5 subtype Avian influenza A viruses sequences available in GenBank,a pair of primers and a TaqMan probe were designed for development of a TaqMan quantitative real-time RT-PCR (RRT-PCR) detecting H5 Avian influenza A Virus. The detection limit of RRT-PCR was 21 plasmid copies. H5N1 and H5N2 Avian influenza A viruses could be identified rapidly. Unrelated RNA samples were not detected. Fine reproducibility was obtained to detect positive plasmid DNA with intra-assay of 2.01% and inter-assay of 4.93%. Beside the way which had the advantage of fast testing speed,it only needed 4 hours from the sample treatment to result report.

Based on the conserved regions of HA and NA genes of H5N1 AIV,two pairs of specific primers were designed and a duplex SYBR Green Ⅰ-based real-time RT-PCR was developed.This method was able to determine AIV H5N1 subtype by detection of H5 and N1 gene in a single tube based on melting temperatures(Tm)discriminations.Two melting peaks at temperatures(79.5±0.3)℃ and(83.3±0.3)℃ represent H5 and N1 gene products,respectively.The PCR products of 150 bp and 474 bp were observed without primer-dimer in agarose gel electrophoresis and confirmed by sequencing.All H5N1 strains tested were positive with two specific melting peaks while H5N2 strains showed a single specific peak of H5 gene.The other subtypes of AIV and viruses yielded no amplification products.The sensitivity of the assay was 21.0 copies·μL-1 for H5 gene recombinant plasmid and 19.5 copies·μL-1 for N1 gene recombinant plasmid,demonstrating 100 and 1 000-fold more sensitive than the conventional RT-PCR method using the same primers.In conclusion,the duplex SYBR Green Ⅰ-based real-time RT-PCR developed in this study can be used for the detection of H5N1 subtype AIV.

According to the chicken β-actin gene sequences available in GenBank,a pair of primers was designed for establish a SYBR Green I quantitative real-time PCR method for β-actin gene of chicken.To establish the standard curve,the product of conventional PCR served as a standard. The analysis of melting curve was also carried out.The results show the linear range of Ct value was from 12 to 31 with a good correlation coefficient(r=0.996).The melting curve show a single peak with a 88±0℃ Tm value.The real-time PCR assay developed in this study can detect β-actin in expand range with high efficiency and less time.The assay provide the basis for β-actin gene of chicken as a reference gene in quantitative analysis of mRNA expression.

传染性支气管炎(IB)是由冠状病毒引起的鸡的一种以呼吸道和泌尿生殖道急性病变为特征的高度接触性传染病,目前已发现肾型毒株至少有16个血清型,不同血清型之间交互免疫力差,常出现免疫失败.该病自1987年在我国流行发生以来,死亡率随年龄的不同可达25%～90%,给养鸡业造成极大的损失,目前尚无特效药物.近年来的研究表明,中药具有显著的抗病毒作用,用于病毒病防治,取得了良好的效果,对IB的中药防治研究也取得了令人鼓舞的效果."双黄杀毒退烧颗粒剂"由黄连、黄芩、蛇床子、五倍子、枝子等14味中药组成,具有杀毒、退烧、扶正功效.试验研究了该药物对肾型传染性支气管炎病毒感染鸡胚及雏鸡的保护效果,结果表明其具有较好的抗IBV作用,能有效降低肾脏损伤,制止死亡.

1 临床症状 2005年7月下旬,某波尔山羊种羊场饲养的300余只5月龄左右的波尔山羊突然出现零星发病.1周内先后有8只分布在不同圈舍的羊发病.病羊主要表现为突然发病,躁动不安,四肢强烈划动,磨牙,口水过多,倒地抽搐,有的羊于发病后12h内死亡.死后羊腹部膨大,口鼻流出泡沫状液体,肛门周围可见少量稀粪和黏液,至就诊时共死亡6只.