对虾IHHNV荧光定量PCR检测方法的建立

根据GenBank中对虾传染性皮下和造血器官坏死病毒(IHHNV)保守基因序列(AF218226),设计合成了1对引物和1条TaqMan探针,建立了检测IHHNV的荧光定量PCR技术.将建立的荧光定量PCR检测方法与常规PCR对比.结果显示,所建立的荧光定量PCR方法灵敏度可达2个拷贝,比常规PCR灵敏度高1 000倍.对保存的15份经常规PCR检测为IHHNV阳性的DNA样品进行荧光定量PCR检测,结果都为阳性,检测的病毒含量为2.15×107～4.21×104拷贝/μL.用该方法对不同浓度的样品进行了重复检测,表明该方法具有良好的重复性,可满足IHHNV的临床诊断需要.     

对虾传染性皮下及造血组织坏死病毒(IHHNV) PCR检测方法的建立

对虾传染性皮下及造血组织坏死病毒(IHHNV)病是国际兽疫局(OIE)划定的甲壳类其他重要疾病之一,它分布较广,危害严重,对世界对虾养殖业发展影响重大.本文根据Genbank登录的IHHNV基因序列(AF218266),设计了1对特异性引物,从纯化的IHHNV DNA和感染IHHNV凡纳滨对虾(Litopenaeus vannamei)组织DNA中成功地扩增出产物大小为703bp的DNA片段,该对引物对IHHNV DNA的检测灵敏度为19.85fg(8.83×103病毒拷贝),与健康对虾组织DNA、对虾白斑综合征病毒(WSSV) DNA及对虾肝胰腺细小病毒(HPV) DNA无交叉反应.本方法可快速、灵敏、特异地检测出对虾感染和携带IHHNV状况,为对虾健康养殖、无特定病原(SPF)种群选育及流行病调查提供了有效的检测手段.     

同时检测两种对虾病毒和4种弧菌的同步PCR方法的建立

通过检索、多重比对、分析和筛选GenBank中对虾白斑综合征病毒(WSSV)、传染性皮下和造血器官坏死病毒(IHHNV)、副溶血孤菌、创伤弧菌、哈维氏弧菌和溶藻胶弧菌的基因序列,设计了10对特异性引物,以已知毒株和菌株的DNA为模板进行PCR,均能扩增出与实验设计相符合的DNA片段,对PCR扩增条件进行优化,建立了可同时检测鉴别WSSV、IHHNV、副溶血弧菌、创伤弧菌、哈维氏弧菌和溶藻胶弧菌,并且能同时区分WSSV不同地理毒株的同步PCR方法.研究结果表明,该方法检测特异性好,检测通量大,适合于对虾多种病原的同时检测.     

PCR技术在对虾病毒病检测上的应用及研究进展

聚合酶链式反应PCR具有特异性强、灵敏度高、快速简便及重复性好等优点,多种临床样品均可用PCR进行检测,现已开发出多种PCR新技术,用于生物学科的各个领域。本文对PCR技术的原理及其在动物疫病尤其是对虾病毒病的检测中的应用及研究进展做一综述,以期对对虾病毒病的诊断与防治有所帮助。     

采用OIE标准检测养殖对虾中传染性皮下及造血组织坏死病毒(IHHNV)的PCR检出类型

利用世界动物卫生组织(OIE)推荐的4对引物(389F/R、392F/R、77012F/77353R 和309F/R),通过普通 PCR 方法,对本实验室2011?2012年采集于国内不同地区的对虾样品进行 IHHNV(Infectious hypodermal and hematopoietic necrosis virus)检测,并对国内存在的 IHHNV 检出类型进行初步分析。检测结果显示,在凡纳滨对虾、斑节对虾、中国对虾、宽沟对虾中均检测出了 IHHNV,而在脊尾白对虾中未检出。其中凡纳滨对虾阳性率最高,中国对虾阳性检测率最低。对虾样品2011年阳性率高于2012年,华东地区高于华北、华南两地。此外,根据4对引物的检测结果,得到国内 IHHNV 的4种 PCR 检出类型。     

对虾6种病毒多重PCR检测方法的建立

本研究针对养殖对虾6种病毒,包括白斑综合征病毒(WSSV)、传染性皮下及造血组织坏死病毒(IHHNV)、肝胰腺细小病毒(HPV)、桃拉综合征病毒(TSV)、对虾杆状病毒(BP)和传染性肌肉坏死病毒(IMNV),选择各自的基因分别设计特异性引物和探针,首先进行了单一病毒的PCR验证,在此基础上建立了同时特异性检测6种对虾病毒的多重PCR检测体系。对反应条件进行优化并进行特异性和灵敏度的验证。50μl反应体系,Mg2+的最佳浓度为5mmol/L,ExTaq酶最佳用量为3.75U,反应程序中最佳退火温度为55.5℃。6种病毒之间以及与对虾基因组都存在很好的特异性。最终经试验验证,该系统的检测灵敏度对WSSV可达104拷贝,IHHNV可达102拷贝,HPV可达104拷贝,TSV可达103拷贝,BP可达105拷贝,IMNV可达105拷贝。虽然该多重PCR方法灵敏度不如单一的PCR检测高,但是通过实际样品检测验证了该方法省时、消耗较少,又不失准确性,在实际应用中具有可靠性和应用价值。     

凡纳滨对虾WSSV、IHHNV和CMNV多重PCR检测方法的建立

根据偷死野田村病毒(CMNV)的保守基因序列,设计筛选出1对特异性引物CMN279,利用已公布的凡纳滨对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)特异性引物WSS235和IHHN356,建立了一种同时检测WSSV、IHHNV和CMNV的多重PCR检测方法。收集18种病原、健康对虾组织及WSSV、IHHNV、CMNV阳性病料,开展特异性测试,该方法可以特异性扩增出WSSV、IHHNV、CMNV基因片段,健康对虾肌肉组织、其他18种病原均未扩增出任何片段,特异性强。应用克隆方法制备目的基因质粒,应用连续稀释质粒方法开展灵敏度测试,测定该方法检测灵敏度分别为WSSV 9.74fg、IHHNV7.65fg、CMNV 10fg;与已报道的多重PCR检测灵敏度相比较,检测灵敏度提高10倍。应用本研究建立的多重PCR检测方法与实验室标准检验方法同时进行22个样品检验,多重PCR检验方法的检验结果与实验室标准方法检验结果符合率为100%。上述实验结果表明:本研究建立的多重PCR检验方法具有特异性强、灵敏度高、检验时间短、检验结果准确度高的特点,可用于WSSV、IHHNV和CMNV三种病原的快速检测诊断。     

对虾传染性皮下及造血组织坏死病毒(IHHNV)的流行病学与检测技术研究进展

对虾传染性皮下及造血组织坏死病(IHHN)是国际兽疫局(OIE)划定的甲壳类其他重要疾病之一，它分布广泛，危害严重，对世界对虾养殖业的发展影响重大。随着国际贸易的不断发展，区域间个体的流动有可能造成该病病毒传播。有迹象表明，近几年来中国养殖对虾中已发现IHHNV，且呈流行趋势。目前，国际上已建立起多种标准检测方法以监控疾病的流行。本文就该病毒的病毒特性、感染宿主、传播途径、地理分布及诊断技术等方面的研究现状作一综述，旨为对虾病毒性流行病学调查及疾病监控提供参考。     

双重PCR同时检测对虾白斑综合征病毒(WSSV)和传染性皮下及造血器官坏死病毒(IHHNV)

根据Genbank中对虾白斑病由白斑综合征病毒（WSSV）和传染性皮下及造血器官坏死病毒（IHHNV）的基因序列,设计了两对能分别检测WSSV和IHHNV保守片段基因的特异性引物,而且这两对引物在同一反应体系中可以同时对WSSV和IHHNV的DNA模板进行多重PCR扩增,得到大小分别为110bp（WSSV）和356bp（IHHNV）的扩增条带。对影响PCR反应的主要因素Mg“浓度和退火温度进行了优化,证明当Mg^2＋浓度为2．0～4．0mmd,退火温度为57～59℃时可获得最佳的扩增和检测效果。特异性试验结果表明,这两对引物检测WSSV和IHHNV具有很好的特异性,对其它对虾常见病原的PCR扩增结果均为阴性。敏感性测定结果表明,该反应体系最低能检测100pg的WSSV和IHHNV的DNA模板。临床检测表明,所建立的双重PCR方法可以适用于WSSV和IHHNV的同时检测和鉴别。     

PCR法制备地高辛标记DNA探针斑点杂交检测对虾传染性皮下及造血组织坏死病毒(IHHNV)

利用非放射性标记物地高辛(DIG),通过PCR方法制备了对虾传染性皮下及造血组织坏死病毒(IHHNV)DNA探针,探针长度705bp,标记产量为20ng／μL。通过核酸探针斑点杂交检测方法对此探针特异性及灵敏度进行验证,结果表明,该探针具有较高的灵敏度和较强的特异性,检测IHHNV DNA的检出灵敏度为24．8pg,可检出26．6ng患病对虾组织DNA中的IHHNV,与250．4ng健康虾组织DNA、202．5ng健康虾匀浆液,白斑综合症病毒(WSSV)DNA和肝胰腺细小病毒(HPV)DNA均不发生交叉反应。本方法可应用于健康亲虾、苗种的培育和无特定病原(SPF)对虾种群的选育及IHHNV流行病学调查,并具有较高的应用价值。     

常规PCR、实时定量PCR检测传染性皮下及造血器官坏死病毒的效果分析

对虾传染性皮下及造血器官坏死病毒(IHHNV)可感染世界各地养殖对虾,给对虾养殖业造成严重经济损失。本实验首次采用实时定量PCR法对广西地区的84份凡纳滨对虾样品进行检测,同时以常规PCR检测作对照。实时定量PCR检测阳性率为79·8%,常规PCR检测阳性率为40·5%,表明广西地区养殖的凡纳滨对虾IHHNV的感染率较高。将二者检测均呈阳性的30份样品扩增产物进行序列分析测序,测序结果通过DNA STAR软件包进行分析,并通过NCBI Blast与GenBank中的序列进行比对。结果证明,测定的是IHHNV序列。30份样品的IHHNV序列很保守,可以分为4种类型,仅有两个碱基的位置发生变异。实时定量PCR检测IHHNV,快速、灵敏、准确,特异性好,可以作为检测对虾感染病毒的有效方法。     

用逆转录多聚酶链式反应从凡纳对虾中检出传染性皮下组织和造血器官坏死病毒

传染性皮下组织和造血器官坏死病毒 (ＩＨＨＮＶ) ,是一种细小病毒 ,它能感染所有起源于中胚层和外胚层的对虾组织细胞[1] 。在许多养殖对虾的国家都有ＩＨＨＮＶ ,特别是中美洲国家和地区[2 ] 的对虾养殖业深受其害。随着各国间对虾贸易的急剧增长 ,ＩＨＨＮＶ地理分布范围日益广泛 ,迄今为止 ,已扩散到中国台湾、新加坡、马来西亚、泰国、印度尼西亚、澳大利亚、菲律宾、厄瓜多尔、秘鲁等国家和地区[2 -4] 。红额角对虾 (Ｐｅｎａｅｕｓｓｔｙｌｉｒｏｓｔｒｉｓ)、斑节对虾 (Ｐ .ｍｏｎｏｄｏｎ)、短沟对虾 (Ｐ .ｓｅｍｉｓｕｌｃａｔｕ…     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).     

凡纳滨对虾(Litopenaeus vannamei)传染性皮下及造血组织坏死病毒(IHHNV)及虾肝肠胞虫(EHP)的荧光定量PCR检测

2013年,河北、天津等地区养殖的凡纳滨对虾(Litopenaeus vannamei)育苗期出现死苗、出苗率低的情况,生产上,仔虾个体大小差异较大,造成了严重损失.本研究采用荧光定量PCR方法(Real-time PCR)对天津大港地区采集的108尾凡纳滨对虾仔虾样品进行单尾病原检测.结果显示,传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)和虾肝肠胞虫(Enterocytozoon hepatopenaei,EHP)均有检出.IHHNV阳性检出率100％,每微克对虾组织DNA的病毒拷贝数为103-107,且个体较大的样品(1.2-2.0 cm)携带病毒拷贝数偏高;EHP阳性检出率为49.1％,每微克对虾组织DNA的拷贝数为103-105,且集中于个体较小样品(0.7-1.1 cm).对IHHNV和EHP阳性凡纳滨对虾样品进行生物学体长与病毒载量指数相关性分析,显示IHHNV载量指数与对虾生长速率呈正相关,虾组织IHHNV平均载量达8.51×104 copies/μg DNA,为较高的感染水平;EHP的载量与对虾生长速率呈负相关关系,与较大个体阳性检出率较低相对应,虾组织EHP平均载量达到2.19× 104 copies/μg DNA,为较高的感染水平.由此,该批凡纳滨对虾仔虾患病为IHHNV和EHP的混合感染所致,本研究数据为IHHNV和EHP病原混合感染流行情况及其对养殖育苗期仔虾生长的影响提供科学依据.     

Detection and quantification of Aeromonas schubertii in Channa maculata by TaqMan MGB probe fluorescence real‐time quantitative PCR

Aeromonas schubertii is a major epidemiological agent that threatens cultured snakeheads (Channidae) and has caused great economic losses in fish‐farming industries in China in recent years. In present study, a specific TaqMan minor groove binder (MGB) probe fluorescence real‐time quantitative PCR (qPCR) assay was developed to rapidly detect and quantify A. schubertii. A pair of qPCR primers and a TaqMan MGB probe were selected from the rpoD gene, which were shown to be specific for A. schubertii. A high correlation coefficient (R² = 0.9998) in a standard curve with a 103% efficiency was obtained. Moreover, the qPCR method's detection limit was as low as 18 copies/μl, which was 100 times more sensitive than that of conventional PCR. The detection results for the A. schubertii in pond water and fish tissue were consistent with those of the viable counts. Bacterial load changes detected by qPCR in different tissues of snakeheads infected with A. schubertii showed that the gills and intestines may be the entry for A. schubertii, and the spleen and kidney are major sites for A. schubertii replication. The established method in present study should be a useful tool for the early surveillance and quantitation of A. schubertii.     

TaqMan real-time PCR assay for relative quantification of white spot syndrome virus infection in Penaeus monodon Fabricius exposed to ammonia

White spot disease is caused by a highly virulent pathogen, the white spot syndrome virus (WSSV). The disease is usually triggered by changes in environmental parameters causing severe losses to the shrimp industry. This study was undertaken to quantify the relative WSSV load in shrimp exposed to ammonia, using a TaqMan‐based real‐time PCR, and their subsequent susceptibility to WSSV. Shrimp were exposed to different levels of total ammonia nitrogen (TAN) (8.1, 3.8 and 1.1 mg L^?1) for 10 days and challenged with WSSV by feeding WSSV‐positive shrimp. WSSV was detected simultaneously in haemolymph, gills and pereopods at four hours post‐infection. The TaqMan real‐time PCR assay showed a highly dynamic detection limit that spanned over 6 log₁₀ concentrations of DNA and high reproducibility (standard deviation 0.33–1.42) and small correlation of variability (CV) (1.89–3.85%). Shrimp exposed to ammonia had significantly higher (P < 0.01) WSSV load compared to the positive control, which was not exposed to ammonia. Shrimp exposed to 8.1 mg L^?1 of TAN had the highest (P < 0.01) WSSV load in all three organs in comparison with those exposed to 3.8 and 1.1 mg L^?1 of TAN. However, haemolymph had significantly higher (P < 0.01) viral load compared to the gills and pereopods. Results showed that shrimp exposed to ammonia levels as low as 1.1 mg L^?1 (TAN) had increased susceptibility to WSSV.     

A real‐time PCR for the detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp

Hepatopancreatic parvovirus (HPV) causes a common shrimp disease that occurs in many shrimp farming regions, especially in the Indo Pacific, and infects most of the cultured penaeid species. There are seven geographic HPV isolates known, so a method to detect different HPV types is needed. We developed a sensitive and generic real‐time PCR assay for the detection of HPV. A pair of primers and TaqMan probe based on an HPV sequence obtained from samples of Fenneropenaeus chinensis from Korea were selected, and they were used to amplify a 92 bp DNA fragment. This real‐time PCR was found to be specific to HPV and did not react with other shrimp viruses. A plasmid (pHPV‐2) containing the target HPV sequence was constructed and used for determination of the sensitivity of this assay. The assay could detect a single copy of plasmid DNA, and it was used successfully in finding HPV in shrimp samples from the China‐Yellow Sea region, Taiwan, Korea, Thailand, Madagascar, New Caledonia and Tanzania.     

Development of a quantitative PCR assay for monitoring Streptococcus agalactiae colonization and tissue tropism in experimentally infected tilapia

Streptococcus agalactiae has become one of the most important emerging pathogens in the aquaculture industry and has resulted in large economic losses for tilapia farms in China. In this study, three pairs of specific primers were designed and tested for their specificities and sensitivities in quantitative real‐time polymerase chain reactions (qPCRs) after optimization of the annealing temperature. The primer pair IGS‐s/IGS‐a, which targets the 16S‐23S rRNA intergenic spacer region, was finally chosen, having a detection limit of 8.6 copies of S. agalactiae DNA in a 20 μL reaction mixture. Bacterial tissue tropism was demonstrated by qPCR in Oreochromis niloticus 5 days post‐injection with a virulent S. agalactiae strain. Bacterial loads were detected at the highest level in brain, followed by moderately high levels in kidney, heart, spleen, intestines, and eye. Significantly lower bacterial loads were observed in muscle, gill and liver. In addition, significantly lower bacterial loads were observed in the brain of convalescent O. niloticus 14 days post‐injection with several different S. agalactiae strains. The qPCR for the detection of S. agalactiae developed in this study provides a quantitative tool for investigating bacterial tissue tropism in infected fish, as well as for monitoring bacterial colonization in convalescent fish.