基于TaqMan qPCR检测凡纳滨对虾样品中虾肝肠胞虫并样检测方法的评价
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摘要
对山东海阳和潍坊的2个养殖凡纳滨对虾群体取样,采用TaqManqPCR逐尾检测肝胰腺中的虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)载量,再将提取的DNA样品按5并1(5∶1)、25并1(25∶1)、50并1(50∶1)、100并1(100∶1)和150并1(150∶1)进行并样,检测并样的EHP载量。设定不同临界循环数为假定灵敏度,定性判断各单尾检测阳性及并样组阳性,比较不同并样模式与检测阳性率、诊断灵敏度、诊断特异性等之间的关系以及定量的准确性。结果表明,检测灵敏度过低,会降低高并样率检测的准确性;阳性率在30%以上时,高并样率的检测结果与单样品检测相符性很好;高载量感染的阳性率不低于6.7%时,50∶1以内的并样能准确得出检测结果;低载量感染的阳性率不低于16%时,25∶1以内的并样能得出较好结果;高载量感染的1.3%阳性率和低载量感染的8%阳性率可能导致所有并样出现假阴性结果;各种并样模式均有很好的诊断特异性,50∶1并样的诊断灵敏度与OIE标准推荐的5∶1并样的接近;各并样检测的EHP载量与单样品检测平均值之比在0.27～2.83范围,二者存在极显著相关性,各种并样模式的定量检测结果在数量级水平能大致反映样品的平均EHP载量。本研究为水生动物疫病诊断和流行病学调查的样品检测提供了参考依据。
Two populations of Litopenaeus vannamei from Haiyang and Weifang in Shandong Province were sampled. TaqMan qPCR was used to measure Enterocytozoon hepatopenaei(EHP) in the shrimp hepatopancreas one by one, and then the extracted DNA samples were pooled by 5-pool(5∶1),25-pool(25∶1), 50-pool(50∶1), 100-pool(100∶1), and 150-pool(150∶1) to test the EHP in the pooled samples. The amplification used a special threshold cycle value(C_t) of samples lower/higher than an assumed critical cycle value(C_a), differentiated as an analyzed positive/negative ratio in the single sample test and pooled sample test. The relationship between different pooling modes and the analyzed positive rate, diagnostic sensitivity, diagnostic specificity, and quantitative accuracy were compared. The results showed that the detection for high pooling rate samples could be reduced in very low analytic sensitivity. When the positive rate of individuals in the pooled samples was above 30%, the detection results of the high pooling rate were consistent with that of the single sample detection. If the positive rate of individuals with heavy infections in the pooled sample were not less than 6.7%, satisfying results could be obtained in the sample with a pooling rate below 50∶1; while the positive rate of individuals with light infections in the pooled sample were not less than 16%, the satisfying result could be obtained in the sample with a pooling rate below 25∶1. The pooled samples with a positive rate below 1.3% of individuals with heavy infection, or a positive rate below 8% of individuals with light infection, might lead to false negative results in all pooling rates. All of the pooling modes have good diagnostic specificity.The sample with a 50∶1 pooling rate had a similar diagnostic sensitivity to the sample with a 5∶1 pooling rate, which is the highest pooling rate recommended by the OIE standard. It had a very significant correlation between the mean of the detected EHP load of pooled sample, and the mean of the known EHP load calculated according to the single sample, with a ratio of 0.27～2.83. The quantitative test results of the pooled samples could roughly reflect the average EHP load of the single sample at the order of magnitude. This study provides a reference for sample detection in aquatic animal disease diagnosis, and epidemiological investigations.
Two populations of Litopenaeus vannamei from Haiyang and Weifang in Shandong Province were sampled. TaqMan qPCR was used to measure Enterocytozoon hepatopenaei(EHP) in the shrimp hepatopancreas one by one, and then the extracted DNA samples were pooled by 5-pool(5∶1),25-pool(25∶1), 50-pool(50∶1), 100-pool(100∶1), and 150-pool(150∶1) to test the EHP in the pooled samples. The amplification used a special threshold cycle value(C_t) of samples lower/higher than an assumed critical cycle value(C_a), differentiated as an analyzed positive/negative ratio in the single sample test and pooled sample test. The relationship between different pooling modes and the analyzed positive rate, diagnostic sensitivity, diagnostic specificity, and quantitative accuracy were compared. The results showed that the detection for high pooling rate samples could be reduced in very low analytic sensitivity. When the positive rate of individuals in the pooled samples was above 30%, the detection results of the high pooling rate were consistent with that of the single sample detection. If the positive rate of individuals with heavy infections in the pooled sample were not less than 6.7%, satisfying results could be obtained in the sample with a pooling rate below 50∶1; while the positive rate of individuals with light infections in the pooled sample were not less than 16%, the satisfying result could be obtained in the sample with a pooling rate below 25∶1. The pooled samples with a positive rate below 1.3% of individuals with heavy infection, or a positive rate below 8% of individuals with light infection, might lead to false negative results in all pooling rates. All of the pooling modes have good diagnostic specificity.The sample with a 50∶1 pooling rate had a similar diagnostic sensitivity to the sample with a 5∶1 pooling rate, which is the highest pooling rate recommended by the OIE standard. It had a very significant correlation between the mean of the detected EHP load of pooled sample, and the mean of the known EHP load calculated according to the single sample, with a ratio of 0.27～2.83. The quantitative test results of the pooled samples could roughly reflect the average EHP load of the single sample at the order of magnitude. This study provides a reference for sample detection in aquatic animal disease diagnosis, and epidemiological investigations.
引文
Arnold ME,Mueller-Doblies D,Carrique-Mas JJ,et al.The estimation of pooled-sample sensitivity for detection of Salmonella in turkey flocks.Journal of Applied Microbiology,2009,107(3):936-943
Chayaburakul K,Nash G,Pratanpipat P,et al.Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand.Diseases of Aquatic Organisms,2004,60(2):89-96
FHS.Suggested procedures for the detection and identification of certain finfish and shellfish pathogens.American Fisheries Society,2014,http://www.afs-fhs.org/bluebook/bluebook-index.php
Gu WD.Estimation of vector prevalence by using pool sampling.Chinese Journal of Parasitology and Parasitic Diseases,1998,16(1):29-33[顾卫东.混合样本方法估测媒介感染率.中国寄生虫学与寄生虫病杂志,1998,16(1):29-33]
Kline RL,Brothers TA,Brookmeyer R,et al.Evaluation of human immunodeficiency virus seroprevalence in population surveys using pooled sera.Journal of Clinical Microbiology,1989,27(7):1449-1452
Liu YM,Qiu L,Cheng DY,et al.The relationship of body length and weight in the Litopenaeus vannamei populations detected Enterocytozoon hepatopenaei.Progress in Fishery Sciences,2017,38(4):96-103[刘雅梅,邱亮,程东远,等.检出虾肝肠胞虫(Enterocytozoon hepatopenaei)的凡纳滨对虾(Litopenaeus vannamei)群体的体长和体重关系.渔业科学进展,2017,38(4):96-103]
Liu YM,Qiu L,Sheng AZ,et al.Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR.Journal of Invertebrate Pathology,2018,151:191-196
Liu Z,Zhang QL,Wan XY,et al.Development of real-time PCRassay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates.Progress in Fishery Sciences,2016,37(2):119-126[刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测.渔业科学进展,2016,37(2):119-126]
Mu?oz-Zanzi CA,Johnson WO,Thurmond MC,et al.Pooledsample testing as a herd-screening tool for detection of bovine viral diarrhea virus persistently infected cattle.Journal of Veterinary Diagnostic Investigation,2000,12(3):195-203
Mu?oz-Zanzi C,Thurmond M,Hietala S,et al.Factors affecting sensitivity and specificity of pooled-sample testing for diagnosis of low prevalence infections.Preventive Veterinary Medicine,2006,74(4):309-322
OIE.Manual of diagnostic tests for aquatic animals.2017,http://www.oie.int/international-standard-setting/aquatic-ma nual/access-online/
Ossiander FJ,Wedemeyer G.Computer program for sample sizes required to determine disease incidence in fish populations.Journal of the Fisheries Research Board of Canada,1973,30(9):1383-1384
Rovira A,Cano JP,Mu?oz-Zanzi C.Feasibility of pooled-sample testing for the detection of porcine reproductive and respiratory syndrome virus antibodies on serum samples by ELISA.Veterinary Microbiology,2008,130(1):60-68
Suebsing R,Prombun P,Srisala J,et al.Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei,in penaeid shrimp.Journal of Applied Microbiology,2013,114(5):1254-1263
Tangprasittipap A,Srisala J,Chouwdee S,et al.The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus(Litopenaeus)vannamei.BMC Veterinary Research,2013,9(1):139
Teng HY,Zhang LM,Sun QW,et al.Pooled sampling method under given sensitivity and specificity in improving accuracy of point estimator of population rate.Academic Journal of Second Military Medical University,2011,32(1):72-75[滕海英,张罗漫,孙庆文,等.给定灵敏度和特异度下混合样本方法对提高总体率点估计精度的作用.第二军医大学学报,2011,32(1):72-75]
Tourtip S,Wongtripop S,Stentiford GD,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationships.Journal of Invertebrate Pathology,2009,102(1):21-29
Williams CJ,Moffitt CM.A critique of methods of sampling and reporting pathogens in populations of fish.Journal of Aquatic Animal Health,2001,13(4):300-309
Chayaburakul K,Nash G,Pratanpipat P,et al.Multiple pathogens found in growth-retarded black tiger shrimp Penaeus monodon cultivated in Thailand.Diseases of Aquatic Organisms,2004,60(2):89-96
FHS.Suggested procedures for the detection and identification of certain finfish and shellfish pathogens.American Fisheries Society,2014,http://www.afs-fhs.org/bluebook/bluebook-index.php
Gu WD.Estimation of vector prevalence by using pool sampling.Chinese Journal of Parasitology and Parasitic Diseases,1998,16(1):29-33[顾卫东.混合样本方法估测媒介感染率.中国寄生虫学与寄生虫病杂志,1998,16(1):29-33]
Kline RL,Brothers TA,Brookmeyer R,et al.Evaluation of human immunodeficiency virus seroprevalence in population surveys using pooled sera.Journal of Clinical Microbiology,1989,27(7):1449-1452
Liu YM,Qiu L,Cheng DY,et al.The relationship of body length and weight in the Litopenaeus vannamei populations detected Enterocytozoon hepatopenaei.Progress in Fishery Sciences,2017,38(4):96-103[刘雅梅,邱亮,程东远,等.检出虾肝肠胞虫(Enterocytozoon hepatopenaei)的凡纳滨对虾(Litopenaeus vannamei)群体的体长和体重关系.渔业科学进展,2017,38(4):96-103]
Liu YM,Qiu L,Sheng AZ,et al.Quantitative detection method of Enterocytozoon hepatopenaei using TaqMan probe real-time PCR.Journal of Invertebrate Pathology,2018,151:191-196
Liu Z,Zhang QL,Wan XY,et al.Development of real-time PCRassay for detecting microsporidian Enterocytozoon hepatopenaei and the application in shrimp samples with different growth rates.Progress in Fishery Sciences,2016,37(2):119-126[刘珍,张庆利,万晓媛,等.虾肝肠胞虫(Enterocytozoon hepatopenaei)实时荧光定量PCR检测方法的建立及对虾样品的检测.渔业科学进展,2016,37(2):119-126]
Mu?oz-Zanzi CA,Johnson WO,Thurmond MC,et al.Pooledsample testing as a herd-screening tool for detection of bovine viral diarrhea virus persistently infected cattle.Journal of Veterinary Diagnostic Investigation,2000,12(3):195-203
Mu?oz-Zanzi C,Thurmond M,Hietala S,et al.Factors affecting sensitivity and specificity of pooled-sample testing for diagnosis of low prevalence infections.Preventive Veterinary Medicine,2006,74(4):309-322
OIE.Manual of diagnostic tests for aquatic animals.2017,http://www.oie.int/international-standard-setting/aquatic-ma nual/access-online/
Ossiander FJ,Wedemeyer G.Computer program for sample sizes required to determine disease incidence in fish populations.Journal of the Fisheries Research Board of Canada,1973,30(9):1383-1384
Rovira A,Cano JP,Mu?oz-Zanzi C.Feasibility of pooled-sample testing for the detection of porcine reproductive and respiratory syndrome virus antibodies on serum samples by ELISA.Veterinary Microbiology,2008,130(1):60-68
Suebsing R,Prombun P,Srisala J,et al.Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei,in penaeid shrimp.Journal of Applied Microbiology,2013,114(5):1254-1263
Tangprasittipap A,Srisala J,Chouwdee S,et al.The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp Penaeus(Litopenaeus)vannamei.BMC Veterinary Research,2013,9(1):139
Teng HY,Zhang LM,Sun QW,et al.Pooled sampling method under given sensitivity and specificity in improving accuracy of point estimator of population rate.Academic Journal of Second Military Medical University,2011,32(1):72-75[滕海英,张罗漫,孙庆文,等.给定灵敏度和特异度下混合样本方法对提高总体率点估计精度的作用.第二军医大学学报,2011,32(1):72-75]
Tourtip S,Wongtripop S,Stentiford GD,et al.Enterocytozoon hepatopenaei sp.nov.(Microsporida:Enterocytozoonidae),a parasite of the black tiger shrimp Penaeus monodon(Decapoda:Penaeidae):Fine structure and phylogenetic relationships.Journal of Invertebrate Pathology,2009,102(1):21-29
Williams CJ,Moffitt CM.A critique of methods of sampling and reporting pathogens in populations of fish.Journal of Aquatic Animal Health,2001,13(4):300-309