栉孔扇贝急性病毒性坏死病毒基因组全序列的测定和核酸诊断技术的研究
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摘要
栉孔扇贝(Chlamys farreri)作为我国北方沿海特有的优良养殖贝类，形成规模化养殖已有20多年的历史，给我国北方沿海地区带来了巨大的经济效益，在我国海水养殖业中占有重要的地位。自20世纪90年代以来，栉孔扇贝的大规模死亡在我国北方养殖海区连年暴发(累积死亡率高达90％以上)，已给栉孔扇贝养殖业造成了巨大的经济损失，并严重阻碍了这一产业的持续发展。
急性病毒性坏死病毒(Acute Viral Necrobiotic Virus，AVNV)，是引起我国北方沿海养殖栉孔扇贝夏季大规模死亡(急性病毒性坏死症)(Acute ViralNecrobiotic Disease，AVND)的病原。该病毒主要在扇贝的消化腺、外套膜、肾脏和肠等的结缔组织细胞和间质细胞中发现，病毒呈球形，直径大小为130～170nm，核衣壳直径为90～140nm，囊膜厚度为5～10nm，外具放射状纤突，纤突长度约为20nm，病毒核衣壳与囊膜间有10～16nm的透明间隙，病毒核心区电子密度较高。这些形态学方面的特征与此前国外报道的感染牡蛎等双壳贝类的牡蛎疱疹病毒(Ostreid Herpesvirus 1，OsHV-1)很相似。
首先应用扩增OsHV-1核酸序列的引物(A3／A4、C2／C6和Gp3／Gp4)，以AVNV核酸为模板进行了PCR扩增，得到了部分AVNV的基因序列。序列分析结果显示，AVNV和OsHV-1在A3／A4、C2／C6和Gp3／Gp4片段的核苷酸相似性分别达到了99％、96％和99％，说明AVNV和OsHV-1在核苷酸水平具有很大的相似性。根据这些已有的核苷酸序列和已经公布的OsHV-1的全基因组序列(GeneBank号为AY509253)设计PCR引物，运用PCR扩增和分子克隆等技术，建立了一系列末端相互重叠的克隆，覆盖整个AVNV的基因组，对这些克隆进行了测序和拼接，完成了AVNV基因组核苷酸序列的测定(GeneBank号：GQ153938)。AVNV基因组全长210，993bp，A和T含量丰富，占61．5％。分析发现，AVNV基因组的两个末端存在较大的反向重复序列(1～7，638和178，052～185，689，187，200～197，411和200，782～210，993)，符合疱疹病毒基因组的基本特征。预测AVNV基因组中含有123个潜在的开放阅读框(Open Reading Frames，ORF)，编码从41个氨基酸残基至1878个氨基酸残基的蛋白质。通过与GeneBank／EMBL／DDBJ数据库中的核酸和蛋白质序列的同源性分析发现，AVNV中有44个ORF具有一定的结构和功能，且与其它类群的核酸或蛋白质氨基酸序列具有显著的同源性。其功能与病毒DNA复制、核酸代谢、修饰以及病毒与宿主相互作用等有关，如DNA聚合酶(DNA polymerase)(ORF99)、解旋酶SF2 helicase(ORF66)、引物酶(Primase) (ORF24)、ATPase subunit of DNA-packagingterminase(ORF108)、核糖核苷酸还原酶小亚基(Ribonucleotide reductase smallsubunit) (ORF20)、核糖核苷酸还原酶大亚基(Ribonucleotide reductase largesubunit)(ORF50)、dUTP酶(deoxyuridine triphosphatase，dUTPase)(ORF 27、ORF 33和ORF74)等。对这些主要基因的密码子偏爱性、结构域、同源性和蛋白质二级结构进行了分析，推测了它们可能的功能。分析了AVNV和其它贝类疱疹病毒(主要是OsHV-1)的关系，发现AVNV和OsHV-1的29个ORF编码的蛋白质氨基酸具有100％的同源性，3个具有81～90％的同源性，8个具有40～80％的同源性。对AVNV的分类地位进行了探讨，尽管AVNV和OsHV-1在分子水平的相似程度非常高，二者的亲缘关系很近，但是二者在基因组大小和ORF编码蛋白质的同源性方面存在较大的差异，结合二者在宿主范围、地理分布和流行病学等方面的不同，认为AVNV是疱疹病毒目中Malacoherpesviridae下的一个新种。
在完成AVNV基因组全序列的测定和分析的基础上，针对不同样品分析的要求，建立了AVNV PCR检测方法、巢式PCR检测方法、环介导等温扩增(LAMP)检测方法和荧光实时定量PCR检测方法，为该疾病的快速诊断、致病机理的探讨、分子流行病学调查和传播途径的调查以及预警监测体系的建立提供了重要的技术保障。
PCR检测方法和巢式PCR检测方法，应用Primer premier 5．0设计了一步PCR引物和巢式PCR引物，预计扩增产物大小分别为706bp和314bp。对反应条件进行了优化，该两种检测方法的灵敏度分别为100fg和1fg的AVNV核酸，特异性实验表明该两对引物只与AVNV发生阳性反应，和扇贝组织DNA以及其它水生生物病毒均无交叉反应。该方法灵敏度高、特异性好且可以直接对PCR产物进行测序确认，适合在实验室内对大批量样品进行检测。
环介导等温扩增(LAMP)检测方法，应用http：／／primerexplorer．jp／e／在线软件PrimerExplorer V4设计LAMP引物，其中包括两对外引物AVNV-F3和AVNV-B3，两对内引物AVNV-FIP和AVNV-BIP(引物AVNV-FIP由F2、TTTT连接子和F1c组成，引物AVNV-BIP由B1c、TTTT连接子和B2组成)。对反应条件进行了优化，当反应混合物在64℃孵育60min时，LAMP方法能用于检测AVNV，最低检测灵敏度达到了1fg的AVNV核酸，而且扩增产物可以通过肉眼直接观察，不需要PCR仪和电泳仪等设备。LAMP是一种操作简单、灵敏度高和特异性好的检测方法，在AVNV的简便、快速检测方面具有重要的应用前景。
荧光实时定量PCR检测方法，根据AVNV基因序列，应用Beacon Designer7．0设计了一对能特异性扩增90bp片段的引物和TaqMan探针，并对反应条件进行了优化。该方法在10~8～10~2病毒拷贝范围内有较好的线性关系，AVNV拷贝数(X)和循环数(C_t)的相关关系为：lgX=-0．29C_t+13．28(相关系数R~2=0．998)，检测AVNV的最低灵敏度为10~2病毒拷贝，特异性实验表明只对AVNV基因组呈阳性反应。该方法可以检测到组织中极微量AVNV核酸，且可以进行定量分析，为研究AVNV的动态变化、病毒载量与AVND暴发的相关性、疾病暴发前病毒的危险临界值以及病毒致病机理等方面的研究提供有力的工具。
The scallop Chlamys farreri is one of the major species cultured in North China and its culture in commercial scale has been performed more than 20 years. However, the great expansion and intensification have induced the occurrence of disease since 1990's, especially the disease called "Acute Viral Necrobioitic Disease" (AVND) has been becoming epizootics since 1997 in north coast of China. The cumulative mortality could be more than 90% and the disease caused by a virus called "Acute Viral Necrobiotic Virus" (AVNV) has been becoming the major limiting factor in the development of the scallop industry, striking the economic process in the north coastal region of China.
The virus particles take the shape of sphere and have a bilaminal envelope with spike outside it. The diameter of virions is approximately 130-170nm, while the nucleocapsids are 90-140nm. The space between the envelope and capsid is about 10-16nm. The virions could be found in the connective tissue cells and interstitial cells of digestive gland, kidney, mantle, and intestine. These morphological characteristics of AVNV are similar to Ostreid herpesvirus 1 (OsHV-1) which was reported to infect other bivalve mollusk such as oyster, clam and scallop.
Firstly, we obtained some nucleic acid sequences of AVNV by using the primers (A3/A4, C2/C6 and Gp3/Gp4) for detection of OsHV-1 and AVNV DNA as DNA template. Analysis of these sequences revealed that the similarity on the A3/A4, C2/C6 and Gp3/Gp4 fragments arrived at 99%, 96% and 99% respectively between AVNV and OsHV-1. It is believed that great similarity is possible at the level of nucleotide between AVNV and OsHV-1. Then, primers for amplification of AVNV nucleotide sequence were designed according to the sequences of AVNV and OsHV-1 complete genome sequence which had been published (AY509253). PCR and molecular cloning techniques were used to set up a series of overlapping clones at the ends, covering the whole genomic sequence of AVNV. The entire genome of AVNV was determined, following the sequencing and splicing (GeneBank accession number: GQ153938). The genome was a linear double-stranded DNA of 210,993bp in length with a base composition of 61.5% A+T. There were two large inverted repeats at the end of AVNV genome (1-7,638 and 178,052-185,689, 187,200-197,411 and 200,782-210,993), which is the typical characteristic of herpesvirus. 123 open reading frames (ORFs) were identified with coding capacity for polypeptides ranging from 41 to 1878 amino acids. The percent coding density of AVNV accounted for 82% of the genetic information in the AVNV genome and the average length of each ORF in the AVNV genome was l,254bp. Computer-assisted analyses of the deduced amino acid sequences revealed that 44 putative gene products showed significant homology to functionally characterized proteins of other species in the Gene Bank/EMBL/GGBJ database. These proteins included enzymes and structural proteins involved in virus replication, nucleotide metabolism and modification and virus-host interaction, such as DNA polymerase (ORF99), ATPase subunit of DNA-packaging terminase (ORF108), SF2 helicase (ORF66), two subunits of ribonucleotide reductase (ORF20 and ORF50), a tentative primase (ORF24), deoxyuridine triphosphatase(ORF74), and dUTPase (ORF27, ORF33 and ORF74). Moreover, an analysis of codon preference, putative conserved domains or signatures, homology and possible function was done on these important genes. In comparison with the ORFs between AVNV and OsHV-1, the two viruses shared 29, 75, 3 and 8 genes with 100%, 91-99%, 81-90%, 40-80% amino acid identity, respectively. Although AVNV is closely related to OsHV-1, there are some divergences between their genomes in size, identity of gene products, host specificity and geological distribution. These differences suggest that AVNV and OsHV-1 shouldn't belong to the same species and AVNV should be considered a separate species belonging to Malacoherpesviridae in herpesvirales.
With the acquisition of AVNV genomic DNA sequence, studies on detection and diagnosis methods of nucleic acid for AVNV were done. These methods included PCR, nested PCR, loop-mediated isothermal amplification (LAMP), and fluorescence quantitative polymerase chain reaction (FQ-PCR). These methods would be very critical for further research on AVNV, such as rapid detection of AVNV suspected case, pathogenic mechanism of AVNV infection, investigation of molecular epidemiology and the relationship between AVNV and outbreak of the disease.
The primer sets for one-step PCR and nested PCR were designed from the DNA sequence of AVNV. The sizes of amplified products were found in each methods coincided with the predicted sizes of 706bp and 314bp respectively, whereas the uninfected scallop DNA, other DNA viruses and control reaction (no DNA template) were negative. The one-step PCR assay was able to detect 100fg AVNV DNA. The sensitivity of nested PCR amplification was 100 times higher than that of the one-step PCR, which corresponded to about 1fg AVNV DNA. These methods will be very useful for sensitive and specific detection of AVNV in the laboratory.
LAMP primers were designed from AVNV genomic DNA sequence following the method of Notomi et al. (2000) and using the LAMP primer designing software PrimerExplorer V4 (http://primerexplorer.jp/e/). A set of four primers recognizing six distinct regions in the target sequence (F1c, F2, F3, B1c, B2, and B3) were designed. Forward inner primer for AVNV (AVNV-FIP) consisted of F1c, a TTTT linker and F2, and backward inner primer (AVNV-BIP) consisted of B1c, a TTTT linker and B2. The two outer primers, AVNV-F3 and AVNV-B3, were located outside the F2-B2 region, respectively. Reaction was carried out in a water bath and reaction temperature and time were optimized at 64°C for 60 min. The lower detection limit of this assay was 1fg AVNV DNA, which was demonstrated 100 times higher than that of one-strp PCR assay. This assay is a simple, rapid, sensitive and specific LAMP technique for detection of AVNV. The visual inspections of LAMP products by the naked-eye require very simple equipments and may facilitate the field application of the assay.
A pair of specific primers which could amplify a 90bp fragment and a fluorescent TaqMan probe was designed, basing on the DNA sequences of AVNV. Fluorescence based quantitative polymerase chain reaction (FQ-PCR) assay for detection of AVNV was developed and optimized. It could obtain excellent linearity when the AVNV genome was between 10~8 and 10~2 copies, lgX = -0.29Ct + 13.28 (correlation coefficient R = 0.998). The detection limit of this assay was about 10 copies. It was confirmed that the FQ-PCR assay was a powerful tool for the detection of AVNV with rapidity, sensitivity, specificity, and quantification. It would be used for further research on AVNV, such as pathogenic mechanism of AVNV infection, dynamic distribution of AVNV, and the relationship between AVNV and outbreak of the disease.
急性病毒性坏死病毒(Acute Viral Necrobiotic Virus，AVNV)，是引起我国北方沿海养殖栉孔扇贝夏季大规模死亡(急性病毒性坏死症)(Acute ViralNecrobiotic Disease，AVND)的病原。该病毒主要在扇贝的消化腺、外套膜、肾脏和肠等的结缔组织细胞和间质细胞中发现，病毒呈球形，直径大小为130～170nm，核衣壳直径为90～140nm，囊膜厚度为5～10nm，外具放射状纤突，纤突长度约为20nm，病毒核衣壳与囊膜间有10～16nm的透明间隙，病毒核心区电子密度较高。这些形态学方面的特征与此前国外报道的感染牡蛎等双壳贝类的牡蛎疱疹病毒(Ostreid Herpesvirus 1，OsHV-1)很相似。
首先应用扩增OsHV-1核酸序列的引物(A3／A4、C2／C6和Gp3／Gp4)，以AVNV核酸为模板进行了PCR扩增，得到了部分AVNV的基因序列。序列分析结果显示，AVNV和OsHV-1在A3／A4、C2／C6和Gp3／Gp4片段的核苷酸相似性分别达到了99％、96％和99％，说明AVNV和OsHV-1在核苷酸水平具有很大的相似性。根据这些已有的核苷酸序列和已经公布的OsHV-1的全基因组序列(GeneBank号为AY509253)设计PCR引物，运用PCR扩增和分子克隆等技术，建立了一系列末端相互重叠的克隆，覆盖整个AVNV的基因组，对这些克隆进行了测序和拼接，完成了AVNV基因组核苷酸序列的测定(GeneBank号：GQ153938)。AVNV基因组全长210，993bp，A和T含量丰富，占61．5％。分析发现，AVNV基因组的两个末端存在较大的反向重复序列(1～7，638和178，052～185，689，187，200～197，411和200，782～210，993)，符合疱疹病毒基因组的基本特征。预测AVNV基因组中含有123个潜在的开放阅读框(Open Reading Frames，ORF)，编码从41个氨基酸残基至1878个氨基酸残基的蛋白质。通过与GeneBank／EMBL／DDBJ数据库中的核酸和蛋白质序列的同源性分析发现，AVNV中有44个ORF具有一定的结构和功能，且与其它类群的核酸或蛋白质氨基酸序列具有显著的同源性。其功能与病毒DNA复制、核酸代谢、修饰以及病毒与宿主相互作用等有关，如DNA聚合酶(DNA polymerase)(ORF99)、解旋酶SF2 helicase(ORF66)、引物酶(Primase) (ORF24)、ATPase subunit of DNA-packagingterminase(ORF108)、核糖核苷酸还原酶小亚基(Ribonucleotide reductase smallsubunit) (ORF20)、核糖核苷酸还原酶大亚基(Ribonucleotide reductase largesubunit)(ORF50)、dUTP酶(deoxyuridine triphosphatase，dUTPase)(ORF 27、ORF 33和ORF74)等。对这些主要基因的密码子偏爱性、结构域、同源性和蛋白质二级结构进行了分析，推测了它们可能的功能。分析了AVNV和其它贝类疱疹病毒(主要是OsHV-1)的关系，发现AVNV和OsHV-1的29个ORF编码的蛋白质氨基酸具有100％的同源性，3个具有81～90％的同源性，8个具有40～80％的同源性。对AVNV的分类地位进行了探讨，尽管AVNV和OsHV-1在分子水平的相似程度非常高，二者的亲缘关系很近，但是二者在基因组大小和ORF编码蛋白质的同源性方面存在较大的差异，结合二者在宿主范围、地理分布和流行病学等方面的不同，认为AVNV是疱疹病毒目中Malacoherpesviridae下的一个新种。
在完成AVNV基因组全序列的测定和分析的基础上，针对不同样品分析的要求，建立了AVNV PCR检测方法、巢式PCR检测方法、环介导等温扩增(LAMP)检测方法和荧光实时定量PCR检测方法，为该疾病的快速诊断、致病机理的探讨、分子流行病学调查和传播途径的调查以及预警监测体系的建立提供了重要的技术保障。
PCR检测方法和巢式PCR检测方法，应用Primer premier 5．0设计了一步PCR引物和巢式PCR引物，预计扩增产物大小分别为706bp和314bp。对反应条件进行了优化，该两种检测方法的灵敏度分别为100fg和1fg的AVNV核酸，特异性实验表明该两对引物只与AVNV发生阳性反应，和扇贝组织DNA以及其它水生生物病毒均无交叉反应。该方法灵敏度高、特异性好且可以直接对PCR产物进行测序确认，适合在实验室内对大批量样品进行检测。
环介导等温扩增(LAMP)检测方法，应用http：／／primerexplorer．jp／e／在线软件PrimerExplorer V4设计LAMP引物，其中包括两对外引物AVNV-F3和AVNV-B3，两对内引物AVNV-FIP和AVNV-BIP(引物AVNV-FIP由F2、TTTT连接子和F1c组成，引物AVNV-BIP由B1c、TTTT连接子和B2组成)。对反应条件进行了优化，当反应混合物在64℃孵育60min时，LAMP方法能用于检测AVNV，最低检测灵敏度达到了1fg的AVNV核酸，而且扩增产物可以通过肉眼直接观察，不需要PCR仪和电泳仪等设备。LAMP是一种操作简单、灵敏度高和特异性好的检测方法，在AVNV的简便、快速检测方面具有重要的应用前景。
荧光实时定量PCR检测方法，根据AVNV基因序列，应用Beacon Designer7．0设计了一对能特异性扩增90bp片段的引物和TaqMan探针，并对反应条件进行了优化。该方法在10~8～10~2病毒拷贝范围内有较好的线性关系，AVNV拷贝数(X)和循环数(C_t)的相关关系为：lgX=-0．29C_t+13．28(相关系数R~2=0．998)，检测AVNV的最低灵敏度为10~2病毒拷贝，特异性实验表明只对AVNV基因组呈阳性反应。该方法可以检测到组织中极微量AVNV核酸，且可以进行定量分析，为研究AVNV的动态变化、病毒载量与AVND暴发的相关性、疾病暴发前病毒的危险临界值以及病毒致病机理等方面的研究提供有力的工具。
The scallop Chlamys farreri is one of the major species cultured in North China and its culture in commercial scale has been performed more than 20 years. However, the great expansion and intensification have induced the occurrence of disease since 1990's, especially the disease called "Acute Viral Necrobioitic Disease" (AVND) has been becoming epizootics since 1997 in north coast of China. The cumulative mortality could be more than 90% and the disease caused by a virus called "Acute Viral Necrobiotic Virus" (AVNV) has been becoming the major limiting factor in the development of the scallop industry, striking the economic process in the north coastal region of China.
The virus particles take the shape of sphere and have a bilaminal envelope with spike outside it. The diameter of virions is approximately 130-170nm, while the nucleocapsids are 90-140nm. The space between the envelope and capsid is about 10-16nm. The virions could be found in the connective tissue cells and interstitial cells of digestive gland, kidney, mantle, and intestine. These morphological characteristics of AVNV are similar to Ostreid herpesvirus 1 (OsHV-1) which was reported to infect other bivalve mollusk such as oyster, clam and scallop.
Firstly, we obtained some nucleic acid sequences of AVNV by using the primers (A3/A4, C2/C6 and Gp3/Gp4) for detection of OsHV-1 and AVNV DNA as DNA template. Analysis of these sequences revealed that the similarity on the A3/A4, C2/C6 and Gp3/Gp4 fragments arrived at 99%, 96% and 99% respectively between AVNV and OsHV-1. It is believed that great similarity is possible at the level of nucleotide between AVNV and OsHV-1. Then, primers for amplification of AVNV nucleotide sequence were designed according to the sequences of AVNV and OsHV-1 complete genome sequence which had been published (AY509253). PCR and molecular cloning techniques were used to set up a series of overlapping clones at the ends, covering the whole genomic sequence of AVNV. The entire genome of AVNV was determined, following the sequencing and splicing (GeneBank accession number: GQ153938). The genome was a linear double-stranded DNA of 210,993bp in length with a base composition of 61.5% A+T. There were two large inverted repeats at the end of AVNV genome (1-7,638 and 178,052-185,689, 187,200-197,411 and 200,782-210,993), which is the typical characteristic of herpesvirus. 123 open reading frames (ORFs) were identified with coding capacity for polypeptides ranging from 41 to 1878 amino acids. The percent coding density of AVNV accounted for 82% of the genetic information in the AVNV genome and the average length of each ORF in the AVNV genome was l,254bp. Computer-assisted analyses of the deduced amino acid sequences revealed that 44 putative gene products showed significant homology to functionally characterized proteins of other species in the Gene Bank/EMBL/GGBJ database. These proteins included enzymes and structural proteins involved in virus replication, nucleotide metabolism and modification and virus-host interaction, such as DNA polymerase (ORF99), ATPase subunit of DNA-packaging terminase (ORF108), SF2 helicase (ORF66), two subunits of ribonucleotide reductase (ORF20 and ORF50), a tentative primase (ORF24), deoxyuridine triphosphatase(ORF74), and dUTPase (ORF27, ORF33 and ORF74). Moreover, an analysis of codon preference, putative conserved domains or signatures, homology and possible function was done on these important genes. In comparison with the ORFs between AVNV and OsHV-1, the two viruses shared 29, 75, 3 and 8 genes with 100%, 91-99%, 81-90%, 40-80% amino acid identity, respectively. Although AVNV is closely related to OsHV-1, there are some divergences between their genomes in size, identity of gene products, host specificity and geological distribution. These differences suggest that AVNV and OsHV-1 shouldn't belong to the same species and AVNV should be considered a separate species belonging to Malacoherpesviridae in herpesvirales.
With the acquisition of AVNV genomic DNA sequence, studies on detection and diagnosis methods of nucleic acid for AVNV were done. These methods included PCR, nested PCR, loop-mediated isothermal amplification (LAMP), and fluorescence quantitative polymerase chain reaction (FQ-PCR). These methods would be very critical for further research on AVNV, such as rapid detection of AVNV suspected case, pathogenic mechanism of AVNV infection, investigation of molecular epidemiology and the relationship between AVNV and outbreak of the disease.
The primer sets for one-step PCR and nested PCR were designed from the DNA sequence of AVNV. The sizes of amplified products were found in each methods coincided with the predicted sizes of 706bp and 314bp respectively, whereas the uninfected scallop DNA, other DNA viruses and control reaction (no DNA template) were negative. The one-step PCR assay was able to detect 100fg AVNV DNA. The sensitivity of nested PCR amplification was 100 times higher than that of the one-step PCR, which corresponded to about 1fg AVNV DNA. These methods will be very useful for sensitive and specific detection of AVNV in the laboratory.
LAMP primers were designed from AVNV genomic DNA sequence following the method of Notomi et al. (2000) and using the LAMP primer designing software PrimerExplorer V4 (http://primerexplorer.jp/e/). A set of four primers recognizing six distinct regions in the target sequence (F1c, F2, F3, B1c, B2, and B3) were designed. Forward inner primer for AVNV (AVNV-FIP) consisted of F1c, a TTTT linker and F2, and backward inner primer (AVNV-BIP) consisted of B1c, a TTTT linker and B2. The two outer primers, AVNV-F3 and AVNV-B3, were located outside the F2-B2 region, respectively. Reaction was carried out in a water bath and reaction temperature and time were optimized at 64°C for 60 min. The lower detection limit of this assay was 1fg AVNV DNA, which was demonstrated 100 times higher than that of one-strp PCR assay. This assay is a simple, rapid, sensitive and specific LAMP technique for detection of AVNV. The visual inspections of LAMP products by the naked-eye require very simple equipments and may facilitate the field application of the assay.
A pair of specific primers which could amplify a 90bp fragment and a fluorescent TaqMan probe was designed, basing on the DNA sequences of AVNV. Fluorescence based quantitative polymerase chain reaction (FQ-PCR) assay for detection of AVNV was developed and optimized. It could obtain excellent linearity when the AVNV genome was between 10~8 and 10~2 copies, lgX = -0.29Ct + 13.28 (correlation coefficient R = 0.998). The detection limit of this assay was about 10 copies. It was confirmed that the FQ-PCR assay was a powerful tool for the detection of AVNV with rapidity, sensitivity, specificity, and quantification. It would be used for further research on AVNV, such as pathogenic mechanism of AVNV infection, dynamic distribution of AVNV, and the relationship between AVNV and outbreak of the disease.
引文
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