猪轮状病毒实时定量荧光PCR检测方法的建立
摘要
猪轮状病毒(Porcine Rotavirus,PRV)属于呼肠孤病毒科、轮状病毒属,它作为腹泻的主要病原在世界范围内广泛流行,是引起仔猪病毒性腹泻的主要病原之一。该病幼畜最为易感,1～4周龄仔猪发病率超过80%,死亡率7～20%,且症状严重,主要表现为严重腹泻,部分病例因严重脱水、酸碱平衡失调、继发感染而死亡。鉴于其危害严重且没有有效治疗手段,所以对于轮状病毒的提前诊断预防尤其重要。在轮状病毒的检测技术中,实时荧光定量PCR(real time fluorescent quantitative PCR,real time FQ-PCR)大多都以SYBR GreenⅠ染料提供荧光信号,能够有效解决常规PCR的污染问题,特异性更强,自动化程度更高,可满足对多种样本检测的需要,已经得到广泛的认可。本次实验先从甘肃某猪场发生腹泻的猪群中采集粪便样品,用恒河猴胚肾细胞(MA-104细胞系)培养,并扩增了PRV,再根据Genebank上已提交的多株猪轮状病毒VP6基因编码序列,利用primers premier 5.0软件设计引物。对采集的样品进行处理及病毒RNA抽提,以Oligo(dT)18为反转录的引物,将RNA反转录为cDNA,进而以设计的一对引物及合适的条件进行PCR扩增,目的是建立一种快速、准确、特异的PRV real time FQ-PCR检测方法。本实验对细胞、病毒做了扩增培养,成功提取出病毒RNA并将其反转录成cDNA,扩增出长1100bp左右的基因片段,初步建立了荧光定量检测方法,根据最后得到荧光定量PCR图,看出阴性样品与空白对照的荧光曲线均为直线,而阳性样品荧光曲线则有很明显的上升,且曲线上升高低与模板浓度呈正比,且可初见“S”型曲线。本次试验为今后进行PRV分子流行病学研究、分子诊断试剂的开发以及基因工程疫苗的研制提供了一些理论依据和物质基础。
Porcine Rotavirus(PRV) belongs to the family reoviridae,species rotavirus.It is one of the major pathogens that cause life-threatening diarrhea in piglets.The young animals are the most easily infected.Its infectious ratio is over 80%,and its death rate is 7～20% in 1～4 weekage pigs.Rotavirus diarrhea’s typically clinical manifestation is serious diarrhea.Partial infected piglets were dead due to severe loss of water,imbalance of acid alkali and secondary infection.accurate diagnosis and effective vaccination are main means for the prevention of porcine diarrhea, so,early diagnosis for the prevention of rotavirus are particularly important.In this study,porcine rotavirus was successfully isolated from diarrheal piglet fecal samples from Gansu province,and PRV was propagated on MA-104 monolayer cell. accordance with the porcine rotavirus strains vp6 gene coding sequence which has been submitted in Genebank, and in accordance with this sequence,using primers premier 5.0 software design Primer. Samples collected on the treatment and viral RNA extraction, take the Oligo (dT)18 as primers in RT-PCR, Reverse transcription of RNA to cDNA, Then to design a pair of primers and suitable conditions for PCR amplification, approximately 1100bp amplified fragment of the gene.To develop a more sensitive and specific diagnostic diagnostic test for rotavirus by applying real-time quantitative RT-PCR technology is our objectives.however,the majority of which reported to date have been used in a quanlitative format.Real-time PCR using SYBR Green I dye as fluorimetry it has proved it self a valuable tool in true quantitation of different taeget nucleic acids at ptesent,and it has engendered wider acceptance of the PCR due to its improved rapidity,sensitivity,reproducibility and the reduced risk of carry-over contamination.We get a table in the end , Negative control sample and the fluorescence curves are the same straight line, and the fluorescence curve of positive samples, there is a visible rise, and rise in the level with the template curve was directly proportional to the concentration. Positive samples at this time of the curve more smooth, and can be seen an initial "S" curve.This study procides the basis evidence and material basis for PRV molecular epidemiology investigation、research of diagnostic reagent and genetic engineering vaccine.
Porcine Rotavirus(PRV) belongs to the family reoviridae,species rotavirus.It is one of the major pathogens that cause life-threatening diarrhea in piglets.The young animals are the most easily infected.Its infectious ratio is over 80%,and its death rate is 7～20% in 1～4 weekage pigs.Rotavirus diarrhea’s typically clinical manifestation is serious diarrhea.Partial infected piglets were dead due to severe loss of water,imbalance of acid alkali and secondary infection.accurate diagnosis and effective vaccination are main means for the prevention of porcine diarrhea, so,early diagnosis for the prevention of rotavirus are particularly important.In this study,porcine rotavirus was successfully isolated from diarrheal piglet fecal samples from Gansu province,and PRV was propagated on MA-104 monolayer cell. accordance with the porcine rotavirus strains vp6 gene coding sequence which has been submitted in Genebank, and in accordance with this sequence,using primers premier 5.0 software design Primer. Samples collected on the treatment and viral RNA extraction, take the Oligo (dT)18 as primers in RT-PCR, Reverse transcription of RNA to cDNA, Then to design a pair of primers and suitable conditions for PCR amplification, approximately 1100bp amplified fragment of the gene.To develop a more sensitive and specific diagnostic diagnostic test for rotavirus by applying real-time quantitative RT-PCR technology is our objectives.however,the majority of which reported to date have been used in a quanlitative format.Real-time PCR using SYBR Green I dye as fluorimetry it has proved it self a valuable tool in true quantitation of different taeget nucleic acids at ptesent,and it has engendered wider acceptance of the PCR due to its improved rapidity,sensitivity,reproducibility and the reduced risk of carry-over contamination.We get a table in the end , Negative control sample and the fluorescence curves are the same straight line, and the fluorescence curve of positive samples, there is a visible rise, and rise in the level with the template curve was directly proportional to the concentration. Positive samples at this time of the curve more smooth, and can be seen an initial "S" curve.This study procides the basis evidence and material basis for PRV molecular epidemiology investigation、research of diagnostic reagent and genetic engineering vaccine.
引文
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