摘要
根据基因库中派琴虫和单孢子虫基因组的保守序列,设计了2对特异性引物和2条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立能够同时检测派琴虫和单孢子虫的二重荧光定量PCR方法。该方法特异性好,对派琴虫和单孢子虫的检测敏感性分别达到400和40个模板拷贝数;此外抗干扰能力强,对派琴虫和单孢子虫不同模板浓度进行组合,仍可有效地同时检测这2个原虫。对广西沿海的49份贻贝病料进行检测,结果派琴虫阳性率为16.3%,检测的派琴虫含量为2.38×106～9.21×102拷贝/μL;提示派琴虫广泛存在于中国南方沿海的养殖贝类中,建立的荧光定量PCR方法可以用于贝类派琴虫的临床快速检测。该方法对单孢子虫检测的敏感性比派琴虫高,而49份贻贝病料未检出单孢子虫,提示需要进行更多临床样品的检测。
A duplex real-time polymerase chain reaction （drt-PCR） assay was developed and optimized to simultaneously detect Perkinsus sp and Haplosporidium sp of shellfish.Two sets of specific oligonucleotide primers for Perkinsus sp and Haplosporidium sp,along with two TaqMan probes specific for each protozoan parasite were used.The drt-PCR results were analyzed using the Light Cycler 2.0 system.Positive and negative samples were used to confirm the sensitivity and specificity of the drt-PCR.The two protozoan parasites were identified and differentiated by the drt-PCR.The sensitivity of the drt-PCR assay was 400 and 40 template copies for Perkinsus sp and Haplosporidium sp,respectively.No positive results （amplified curves） were displayed when nucleic acid from Marteilia refringens,Aeromonas hydrophila,Pseudomonas fluorescens,Vibrio parahaemolyticu,Vibrio Alginolyticu,Vibrio Fluvialis and Vibrio Mimicus were used as PCR templates.Forty nine mussel samples of Guangxi coastal were tested by this drt-PCR,Perkinsus sp positive infection rate was 16.3%.The results suggested that Perkinsus sp existed in the cultivated shellfish in south China.No Haplosporidium sp positive infection was found in forty nine mussel.There is a great need for detecting much more clinical samples to verify wheeher Haplosporidium sp exist in the shellfish.This drt-PCR assay is a quick,sensitive,and specific test for detection of Perkinsus sp and Haplosporidium sp and will be useful for the control of these protozoan parasites in shellfish.
出处
《中国兽医学报》
CAS
CSCD
北大核心
2010年第7期917-921,共5页
Chinese Journal of Veterinary Science
基金
国家百千万人才工程人选专项资金项目(945200603)
广西科技攻关资助项目(桂科攻0630001-3M)
