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1.
对虾WSSV和IHHNV多重PCR检测试剂盒的研究与应用 总被引：1，自引：0，他引：1
在对虾白斑综合征病毒(WSSV)和传染性皮下和造血器官坏死病毒(IHHNV)二温式PCR检测技术前期研究的基础上,将这2种病原的特异性引物与PCR基础反应液一起组装成快速检测试剂盒。通过对试剂盒敏感性、稳定性和保存期等技术指标的测定以及对临床样品的检测试验,该试剂盒对同一样品中的WSSV和IHHNV DNA模板均能特异性地扩增出2条与实验设计相符的593 bp(WSSV)和356 bp(IHHNV)的DNA片段,而对其它对虾病原的扩增结果均为阴性;该试剂盒最低能检测到102pg的WSSV DNA和10 pg的IHHNV DNA。对WSSV和IHHNV DNA的检出量分别为102~103pg和10~102pg。 相似文献
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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 总被引：6，自引：1，他引：6
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. 相似文献
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多重RT-PCR体系检测4种虾病毒的方法 总被引：1，自引：1，他引：0
根据多重RT-PCR的技术原理,利用对虾传染性表皮与造血组织坏死症病毒、白斑综合征病毒、黄头病毒和桃拉综合征病毒的基因序列分别设计了4对特异引物,建立多重RT-PCR体系用于虾4种病毒的检测。多重RT-PCR体系能特异地扩增出IHHNV、WSSV、YHV和TSV的目的片段:TSV特异性扩增片段508 bp,WSSV 特异性扩增片段435 bp,IHHNV 特异性扩增片段301 bp 和YHV。特异性扩增片段614 bp。结果表明,多重PCR虾病毒检测系统具有较高的特异性和敏感性,并对其它对虾病原呈阴性。IHHNV、TSV、WSSV和YHV模板在多重PCR虾病毒检测体系中的检测下限分别为0.1,1,0.02和0.2 pg。病毒感染病料检测试验中,该检测体系的检测结果与单纯PCR的检测结果呈现出较好的吻合度。 相似文献
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二温式PCR检测对虾传染性皮下和造血器官坏死病毒的研究 总被引：2，自引：0，他引：2
根据对虾传染性皮下和造血器官坏死病毒(IHHNV)基因序列,设计了1对可以扩增356 bp IHH-NV某段基因序列的特异性引物,优化建立了能快速检测IHHNV的二温式PCR。特异性试验和敏感性试验结果表明,该技术只对IHHNV DNA模板进行扩增,得到356 bp的DNA扩增片段,而对SPF南美白对虾组织DNA和其它对虾病原DNA/RNA的扩增结果为阴性;该技术最低能检测到10 pg的IHHNV感染对虾组织样品总DNA。应用该技术对400份对虾临床样品进行检测,结果有96份样品呈现阳性,表明该病在我国南方的养殖对虾中广泛存在,二温式PCR技术可以直接用于该病的临床快速检测和流行病学调查。 相似文献
6.
同时检测两种对虾病毒和4种弧菌的同步PCR方法的建立 总被引：3，自引：1，他引：2
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. 相似文献
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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。 相似文献
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分别针对桃拉综合征病毒（TSV）基因、传染性皮下和造血器官坏死病毒（IHHNV）基因,设计了2对特异性引物,通过对多重RT-PCR扩增条件的优化,建立了快速检测TSV和IHHNV的二温式多重RT-PCR技术.特异性和敏感性试验结果表明,该技术只对TSVRNA和IHHNVDNA进行扩增,分别得到1条231bp的TSV特异性cDNA扩增片段和1条356bp的IHHNV特异性DNA扩增片段,对其它对虾病原核酸的扩增结果为阴性,该技术最低能同时检测到10pg的TSVRNA和IHHNVDNA,具有高度的特异性和敏感性.临床应用试验证明,该技术可以用于对虾养殖业中TSV和IHHNV的快速检测和鉴别诊断. 相似文献
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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。 相似文献
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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) 总被引：10，自引：1，他引：10
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。 相似文献
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采用对虾白斑综合症病毒和传染性皮下组织坏死病毒的特异引物,在一个PCR反应中同时扩增到大小分别为271 bp和356 bp的病毒特异片段,多重PCR条件优化后可从最少100 fg级病毒DNA模板中定性检测出此两种病毒。 相似文献
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应用PCR和RT-PCR技术对4种对虾病毒的检测 总被引：5，自引：3，他引：5
探讨了应用PCR和RT－PCR技术对4种主要的对虾病毒进行检测的方法。同时使用该方法检测了对虾天然饵料－－卤虫中的4种对虾病毒。结果显示，含病毒核酸的阳性对照样品分别扩增出了大小为824bp,705bp,260bp和216bp的预期产物，但未能从卤虫样品中检测到此4种病毒的存在。本文报道的病毒检测方法具有快速、灵敏、准确的特点，可以用于进出口贸易中对活体或冰冻对虾，虾苗，对虾饵料等进行相关对虾病毒的检疫，也为制定我国对虾病毒检疫检验规范提供了技术参考。 相似文献
14.
Linda M. Nunan Steve M. Arce Ronald J. Staha Donald V. Lightner 《Journal of the World Aquaculture Society》2001,32(3):330-334
Abstract.— In March 2000, 104 wild caught Litopenaeus wannamei broodstock, captured off the Pacific coast of Panama, were screened for the following penaeid viruses: infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). The purpose of this study was to determine the prevalence of IHHNV and WSSV in wild shrimp in this area of the Western Hemisphere and to acquire specific pathogen free (SPF) L. vannamei for inclusion into the Oceanic Institute's genetic breeding program. The prevalence of the viruses was determined using the dot blot hybridization format, which is a commercially available molecular method for detecting these viruses. Dot blot hybridization assays can be used as an initial screening method to detect moderately to highly infected shrimp. The results from the dot blot assays indicated the prevalence of IHHNV in 28% and WSSV in 2% of the 104 hemolyrnph samples tested. Results from this study were used to establish the initial candidate SPF status of the animals that were assessed and to determine the prevalence of two serious pathogens of penaeid shrimp captured from the wild of the Pacific Ocean in the Central American region off the coast of Panama. 相似文献
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Competition of infectious hypodermal and haematopoietic necrosis virus (IHHNV) with white spot syndrome virus (WSSV) for binding to shrimp cellular membrane 
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下载免费PDF全文 Infectious hypodermal and haematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV) are two widespread shrimp viruses. The interference of IHHNV on WSSV was the first reported case of viral interference that involved crustacean viruses and has been subsequently confirmed. However, the mechanisms underlying the induction of WSSV resistance through IHHNV infection are practically unknown. In this study, the interference mechanisms between IHHNV and WSSV were studied using a competitive ELISA. The binding of WSSV and IHHNV to cellular membrane of Litopenaeus vannamei was examined. The results suggested that there existed a mutual competition between IHHNV and WSSV for binding to receptors present on cellular membrane of L. vannamei and that the inhibitory effects of WSSV towards IHHNV were more distinct than those of IHHNV towards WSSV. 相似文献
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Melena J Bayot B Betancourt I Amano Y Panchana F Alday V Calderón J Stern S Roch P Bonami JR 《Journal of fish diseases》2006,29(10):589-600
Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response. 相似文献
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S. Thamizhvanan S. Sivakumar S. Santhosh Kumar D. Vinoth Kumar S. Suryakodi K. Balaji T. Rajkumar S. Vimal S. Abdul Majeed G. Taju A. S. Sahul Hameed 《Journal of fish diseases》2019,42(3):447-454
White leg shrimp, Penaeus vannamei, were collected on a monthly basis from grow‐out ponds located at Tamil Nadu and Andhra Pradesh states along the east coast of India for screening of viral and other pathogens. Totally 240 shrimp samples randomly collected from 92 farms were screened for white spot syndrome virus (WSSV), infectious hypodermal and haematopoietic necrosis virus (IHHNV), infectious myonecrosis virus (IMNV) and Enterocytozoon hepatopenaei (EHP). The number of shrimp collected from shrimp farms ranged from 6 to 20 based on the body weight of the shrimp. All the shrimp collected from one farm were pooled together for screening for pathogens by PCR assay. Among the samples screened, 28 samples were WSSV‐positive, one positive for IHHNV and 30 samples positive for EHP. Among the positive samples, four samples were found to be positive for both WSSV and EHP, which indicated that the shrimp had multiple infections with WSSV and EHP. This is the first report on the occurrence of multiple infections caused by WSSV and EHP. Multiplex PCR (m‐PCR) protocol was standardized to detect both pathogens simultaneously in single reaction instead of carrying out separate PCR for both pathogens. Using m‐PCR assay, naturally infected shrimp samples collected from field showed two prominent bands of 615 and 510 bp for WSSV and EHP, respectively. 相似文献
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江苏地区对虾3种病毒病的流行病学调查及5株IHHNV的编码区基因序列分析 总被引：1，自引：1，他引：0
对虾白斑综合征病毒(WSSV)、桃拉综合征病毒(TSV)、传染性皮下及造血组织坏死病毒(IHHNV)是威胁对虾养殖的重要病毒。为了调查这3种病在江苏境内的流行情况,根据世界动物卫生组织(OIE)推荐的《水生动物疾病诊断手册》的PCR检测法对2015年5月—2016年5月采集的1436尾对虾样品进行3种疾病的流行病学调查。结果显示WSSV阳性率为17.20%,TSV阳性率为0%,IHHNV阳性率为39.48%。对江苏不同地区分离出的5株IHHNV进行基因序列分析,数据显示这5个地区的毒株均属于Ⅰ型感染株,与韩国株的进化关系较为接近。本研究通过调查以上3种病毒病在江苏境内的流行情况,并进行序列分析,发现江苏不同地区的凡纳滨对虾IHHNV感染均为Ⅰ型,研究结果对养殖虾的疾病防控有重要参考价值。 相似文献
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Praveen Rai Balakrishnan Pradeep Muhammed P. Safeena Iddya Karunasagar Indrani Karunasagar 《Aquaculture (Amsterdam, Netherlands)》2009,295(3-4):168-174
Detection of infectious hypodermal and hematopoietic necrosis virus (IHHNV) in shrimp is complicated by the fact that certain virus-related sequences are integrated into the genome of Penaeus monodon in some parts of the world, which has been reported so far from Africa and Australia. In this study, we evaluated the highly specific and sensitive diagnostic primer sets for detection of infectious IHHNV and integrated virus-related sequence in 177 samples of P. monodon from India. A nested primer set, IHHNV648F/R and IHHNV309F/R was used to specifically detect infectious IHHNV and not the virus-related sequences. IHHNV was detected in 67.4% postlarvae (PL) and 34% adult samples using this primer set. The OIE recommended primers IHHNV392F/R and IHHNV389F/R gave positive reaction for 86.7% PL and 67% adult samples, while the primer pair 77012F and 77353R gave positive reaction with 46.7% PL and 20% adult samples. These primers were found to detect virus-related sequence integrated into the shrimp genome. The analysis of virus-related sequence by MG831F/R primers showed that 33.7% PL and 31.7% adult shrimp possessed Type A virus-related sequence. 22.8% PL and 10.5% adults had both IHHNV and Type A virus-related sequence. Cloning and sequencing 832 bp virus-related sequence from P. monodon from India revealed presence of five shrimp DNA markers between 439 and 825 bp. This study is the first conclusive report on the presence of Type A virus-related sequence in P. monodon from India. 相似文献