帕利亚姆血清群病毒qRT-PCR检测方法的建立与应用

Establishment of a qRT-PCR for the Palyam Serogroup Virus

帕利亚姆血清群病毒(Palyam serogroup virus,PALV)具有多种血清型,可引起感染的妊娠家畜出现生产异常,在我国呈广泛流行的趋势,但目前尚无该病毒的高通量快速检测技术。本研究旨在建立PALV的高通量逆转录实时荧光定量(Real-time quantitative reverse transcription polymerase chain reaction,qRT-PCR)检测方法用于临床样本中病毒的检测。采用一步法RT-PCR对我国流行的PALV代表毒株的基因节段7(Seg-7)进行全长序列测定,根据Seg-7高度保守的区域合成特异性PCR引物与TaqMan探针,建立PALV的qRT-PCR检测方法;以体外转录PALV的Seg-7 ssRNA为模板,分析qRT-PCR检测方法的特异性、敏感性与重复性;以我国不同血清型的PALV分离毒株及血液样本为检测对象,分析qRT-PCR检测方法的实际检测效果。测序结果显示我国流行的不同血清型PALV毒株Seg-7序列高度保守,且与日本毒株的核酸序列相似度为99.59%~99.80%。建立的PALV群特异性qRT-PCR检测方法具有良好的灵敏度、特异性与准确性:可检测PALV最低核酸拷贝数为23拷贝,与蓝舌病病毒、流行性出血病病毒、非洲马瘟病毒、西藏环状病毒、云南环状病毒、广西环状病毒、牛流行热病毒以及阿卡斑病毒之间无交叉反应;对我国不同血清型的PALV分离毒株及分离病毒的血液样本的检测结果吻合率达到100%。对120份临床血液样本进行PALV核酸与抗体检测结果显示:qRT-PCR检测阳性率为35%,而抗体C-ELISA检测阳性率为30%,二者吻合率为85.7%。本研究提示PALV的Seg-7序列高度保守,可作为病毒核酸检测的靶基因。本研究所建立的PALV群特异性qRT-PCR检测方法具有特异性强、敏感性高、可靠性好的优势,为开展PALV诊断与流行病学监测提供了新的技术手段。

The Palyam serogroup virus(PALV)includes multiple serotypes and is prevalent in China. It can cause birth defects in cattle and sheep. However,high-throughput and fast detection methods of PALV are lacking. We wished to establish a method for rapid detection of the PALV in clinical samples. A one-step reverse transcription-polymerase chain reaction(RT-PCR)was used to determine the full-length sequence of genome segment 7(Seg-7)of the representative strain of the PALV prevalent in China. Specific primers and TaqManTM probes were synthesized based on the highly conserved region of Seg-7,and a qRT-PCR of PALV established. Experiments focusing on the specificity,sensitivity and repeatability of the qRT-PCR were undertaken using Seg-7 ssRNA of the PALV transcribed in vitro. To demonstrate the detection ability of the qRT-PCR,PALV isolates of different serotypes and blood samples were employed. The full-length sequence of Seg-7 from different serotypes prevalent in China were conserved and shared 99.59%~99.80% similarity with the nucleic acid sequences of Japanese strains. The established PALV serogroup-specific qRT-PCR possessed good sensitivity,specificity and accuracy. The qRT-PCR showed a limit of detection of 23 copies/μL and did not cross-react with the bluetongue virus,epizootic hemorrhagic disease virus,African horse sickness virus,Tibet orbivirus, Yunnan orbivirus,Guangxi orbivirus, Bovine ephemeral fever virus or Akabane virus.Simultaneously,the qRT-PCR could detect nucleic acids from PALV isolates and clinical anticoagulant samples with a coincidence rate up to 100%. Detection of nucleic acids and antibodies from 120 clinical blood samples using the qRT-PCR and a complement-enzyme linked immunosorbent assay was 35% and 30%,respectively. Our data suggested that the Seg-7 sequence of the PALV was highly conserved and could be used as a target gene for detection of nucleic acids. Our qRT-PCR was specific,sensitive and reliable,and could provide a new means for the diagnosis of PALV infection and epidemiologic monitoring.