CN114592091B - 一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法 - Google Patents
一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法 Download PDFInfo
- Publication number
- CN114592091B CN114592091B CN202210276386.3A CN202210276386A CN114592091B CN 114592091 B CN114592091 B CN 114592091B CN 202210276386 A CN202210276386 A CN 202210276386A CN 114592091 B CN114592091 B CN 114592091B
- Authority
- CN
- China
- Prior art keywords
- primer
- seq
- rhabdovirus
- kit
- probe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241001125889 Micropterus salmoides Species 0.000 title claims description 20
- 238000001514 detection method Methods 0.000 title abstract description 28
- 239000000523 sample Substances 0.000 claims abstract description 31
- 230000003321 amplification Effects 0.000 claims abstract description 13
- 238000003199 nucleic acid amplification method Methods 0.000 claims abstract description 13
- 239000002773 nucleotide Substances 0.000 claims abstract description 12
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 12
- 241000700605 Viruses Species 0.000 claims description 21
- 201000010099 disease Diseases 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- 238000006243 chemical reaction Methods 0.000 claims description 7
- 238000003745 diagnosis Methods 0.000 claims description 7
- 238000003757 reverse transcription PCR Methods 0.000 claims description 6
- 238000000034 method Methods 0.000 claims description 5
- 239000002299 complementary DNA Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 238000000137 annealing Methods 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000004925 denaturation Methods 0.000 claims description 3
- 230000036425 denaturation Effects 0.000 claims description 3
- 239000007850 fluorescent dye Substances 0.000 claims description 3
- 238000010791 quenching Methods 0.000 claims description 3
- 230000000171 quenching effect Effects 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 230000003612 virological effect Effects 0.000 abstract description 5
- 108020004707 nucleic acids Proteins 0.000 abstract description 4
- 150000007523 nucleic acids Chemical class 0.000 abstract description 4
- 102000039446 nucleic acids Human genes 0.000 abstract description 4
- 238000005516 engineering process Methods 0.000 description 10
- 241000251468 Actinopterygii Species 0.000 description 7
- 241000813323 Maize streak Reunion virus Species 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- 241000711931 Rhabdoviridae Species 0.000 description 6
- 239000006148 magnetic separator Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 241000269799 Perca fluviatilis Species 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 244000052769 pathogen Species 0.000 description 4
- 241000238557 Decapoda Species 0.000 description 3
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 3
- 241000696962 White spot syndrome virus Species 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000439574 Decapod penstyldensovirus 1 Species 0.000 description 2
- 241000701372 Iridovirus Species 0.000 description 2
- 238000007397 LAMP assay Methods 0.000 description 2
- 241000986818 Perhabdovirus Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 241000499489 Castor canadensis Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000032969 Hemorrhagic Septicemia Diseases 0.000 description 1
- 206010062023 Intestinal cyst Diseases 0.000 description 1
- 235000011779 Menyanthes trifoliata Nutrition 0.000 description 1
- 241001112535 Novirhabdovirus Species 0.000 description 1
- 108020004711 Nucleic Acid Probes Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- 241001184198 Orthosiphon Species 0.000 description 1
- 208000014645 Pasteurella hemorrhagic septicemia Diseases 0.000 description 1
- 235000006040 Prunus persica var persica Nutrition 0.000 description 1
- 240000006413 Prunus persica var. persica Species 0.000 description 1
- 101100209986 Rattus norvegicus Slc18a1 gene Proteins 0.000 description 1
- 241000404975 Synchiropus splendidus Species 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000002360 explosive Substances 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000009340 pathogen transmission Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 244000052613 viral pathogen Species 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/158—Expression markers
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法,涉及分子生物学技术领域。所述试剂盒包括以下组份:引物对、探针;所述引物对包括正向引物和反向引物,正向引物的核苷酸序列如SEQ ID No.2所示,反向引物的核苷酸序列如SEQ ID No.3所示;所述探针的核苷酸序列如SEQ ID No.1所示。本发明的试剂盒具有极高的特异性,且非常稳定,能有效的避免非特异性扩增而产生的假阳性;具有高灵敏度,对于病毒核酸分子的最低检测极限可达到101拷贝数。本发明的检测方法具有快速高效、操作简便和鉴定简便的优点。
Description
技术领域
本发明涉及分子生物学技术领域,具体涉及一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法。
背景技术
弹状病毒科(Rhabdoviridae)病毒是一类具有广泛宿主的负链RNA病毒,其颗粒呈现子弹状或棒状。该病毒是危害加州鲈鱼、鳜鱼等名优鱼类养殖产业的重大病原之一,能引起鱼类出现严重的出血性败血症。在2007年,国际病毒分类委员会(InternationalCommittee on Taxonomy of Viruses,ICTV)发表了最新的病毒分类第十次报告,将弹状病毒科(Rhabdoviridae)划分为18个属,其中能够感染鱼类的弹状病毒有Perhabdovirus,Sprivivirus和Novirhabdovirus三个属。加州鲈鱼感染的为Perhabdovirus属病毒,该病毒具有很高的传染性和致病性,可以通过亲本垂直传播和通过水源、鱼饵、带毒鱼、鱼卵、寄生虫进行水平传播。尤其是感染该病毒的鱼苗的死亡率可以达到100%,给广大养殖户造成了重大的经济损失。
近几年,受养殖规模扩大、养殖密度升高及池塘老化等因素的影响,加州鲈鱼绿色疾病防控技术的开发逐渐成为该行业健康可持续发展的瓶颈问题。据已有的文献报道,目前加州鲈鱼的主要疾病以病毒性及细菌性疾病为主,其中病毒性病原主要包括弹状病毒(Micropterus salmoides Rhabdovirus,MSRV)和虹彩病毒。加州鲈鱼弹状病毒病为新发的病毒性疾病,该病毒属弹状病毒科,目前已知该病毒在加州鲈鱼的亲鱼及幼苗中有检测发现,且主要在加州鲈鱼的幼苗阶段发病,死亡率高达80%以上,严重时可达100%。携带病毒的苗种很容易在饲养方式改变、投放外塘、天气变化较大等外界因素的影响下,发生爆发性死亡现象,给苗种企业和养殖户带来巨大经济损失。由此可见,提供无病毒的苗种对于提高加州鲈鱼养殖成活率具有非常重要的意义。
病原早期诊断检测在防止病原传播上具有极其重大的意义,目前常用的诊断技术包括聚合酶链式反应技术(Polymerase Chain Reaction,PCR)、核酸探针技术、环介导恒温扩增技术技术(loop-mediated isothermal amplification,LAMP)、酶联免疫吸附技术以及病原培养法检测技术等。这些诊断技术各有优点和适用范围,对检测条件及操作者都有较高的要求。方便、快速的病原检测技术对于水产养殖基层苗种检疫及及时针对性用药具有重要意义。
虽然传统病毒病诊断主要依靠病毒分离,细胞转染、电镜观察以及普通PCR检测等方法,但缺乏可对病毒进行快速、准确、定量分析的检测预警方法。荧光定量PCR方法具有操作简单、快速,不易出现假阳性,随时对扩增产物进行精确定量分析等优点,逐渐应用于病原微生物的检测与研究中。
发明内容
本发明针对加州鲈鱼弹状病毒(Micropterus salmoides Rhabdoviridae,MSRV)的保守区设计引物,建立MSRV的荧光定量PCR检测方法,提供一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法。
本发明所采用的技术方案如下:
本发明提供了一种靶标在检测加州鲈鱼弹状病毒的试剂盒中的应用,所述靶标核苷酸序列如SEQ ID No.1所示。
本发明还提供了一种用于加州鲈鱼弹状病毒检测的寡核苷酸序列组合,包括引物对序列和探针序列,所述引物对序列包括正向引物序列和反向引物序列,正向引物序列如SEQ ID No.2所示,反向引物序列如SEQ ID No.3所示;所述探针序列如SEQ ID No.1所示。
本发明还提供了一种用于加州鲈鱼弹状病毒检测的试剂盒,包括以下组份:(1)引物对;(2)探针;
所述引物对包括正向引物和反向引物,正向引物的核苷酸序列如SEQ ID No.2所示,反向引物的核苷酸序列如SEQ ID No.3所示;所述探针的核苷酸序列如SEQ ID No.1所示。
用于加州鲈鱼弹状病毒检测的试剂盒还包括含有核苷酸序列如SEQ ID No.1所示的靶标的阳性对照。
优选的,所述探针的5’端标记荧光染料FAM,3’端标记淬灭基团BHQ1。
本发明还提供了上述的试剂盒在非疾病诊断为目的的加州鲈鱼弹状病毒检测中的应用。
本发明还提供了一种非疾病诊断为目的的加州鲈鱼弹状病毒的检测方法,包括以下步骤:
(1)提取待测样品病毒RNA,将待测样品病毒RNA逆转录成cDNA;
(2)以步骤(1)中的cDNA作为扩增模板,使用上述的试剂盒进行特异性RT-PCR扩增;
(3)若能够扩增得到信号,说明待检测样品中含有加州鲈鱼弹状病毒。
步骤(2)中RT-PCR反应体系为:5×One Step Buffer 6μL,正向引物和反向引物各为6pmol,探针为3pmol,DNA模板为1-100ng,无菌无核酸酶水补至30μL;扩增反应条件为55℃15min,95℃预变性30s;95℃变性10s,60℃退火30s,共40个循环。
本发明的有益效果:
本发明的试剂盒具有极高的特异性,且非常稳定,能有效的避免非特异性扩增而产生的假阳性;具有高灵敏度,对于病毒核酸分子的最低检测极限可达到101拷贝数。本发明的检测方法具有快速高效、操作简便和鉴定简便的优点,本发明可根据仪器实时读取的荧光信号来判断扩增结果,无需电泳等其他任何分析步骤。
附图说明
图1为阳性对照的标准曲线图。
图2为实施例1中荧光检测结果图。
图3为实施例2中引物特异性实验的结果图。
图4为实施例3中灵敏度实验结果图。
具体实施方式
实验材料:样本1:于2021.06.16在平湖获取的加州鲈鱼鱼苗。样本2:于2021.04.02在杭州获取的加州鲈鱼鱼苗。
实施例1
检测加州鲈鱼弹状病毒。
(1)加州鲈鱼弹状病毒RNA的提取
样本前处理:鱼肉样本0.7g+800μL PBS,匀浆10s,间隔30s,3次循环,然后4℃、10000rpm离心1min,取上清。
提取操作步骤:
a.裂解与结合
取一离心管,加入200μL样本,依次加入40μL Proteinase K,400μL LysisBuffer,1μL Carrier RNA,200μL异丙醇和20μL BeaverBeads(海狸生物,70406),调整合适的转速漩涡振荡混合均匀,将离心管置于55℃加热15min(每隔5min颠倒数次)。然后将离心管置于磁性分离器上静置1min,用移液器吸去上清液。
b.洗涤
(I)加入600μL Washing Buffer I,漩涡震荡10s,使磁珠充分重悬,将离心管置于磁性分离器上直至溶液澄清,用移液器移去上清液并取下离心管。
(II)加入600μL Washing Buffer II,漩涡震荡10s,使磁珠充分重悬,将离心管置于磁性分离器上直至溶液澄清,用移液器移去上清液并取下离心管。
(III)重复步骤(II)一次。
c.干燥
保持离心管于磁性分离器上,将其置于超净工作台中风干至磁珠表面无明显光泽(2~4min)。
d.洗脱
加入30μL Nuclease-free Water,漩涡震荡1min,使磁珠充分重悬,于55℃加热5min后,将离心管置于磁性分离器上直至溶液澄清,转移上清液至新的离心管中,此即为纯化得到的病毒基因组,可保存于-20℃。
(2)引物、探针的设计
根据引物设计原则,进行引物设计,获得一组引物序列和一条探针序列,其核苷酸序列如下所示(安徽通用合成):
MSRV-F(SEQ ID No.2):AGACACCATACATGCCAGAAG
MSRV-R(SEQ ID NO.3):GTTGTACGGGTCCATGAAGAT
MSRV-P(SEQ ID NO.4):5’-TGCGATTGGGCTACAATCAGCCAT-3’;
MSRV-P的5’端标记荧光染料FAM,3’端标记淬灭集团BHQ1。
(3)荧光定量PCR实验
PCR反应体系如表1所示。
表1反应体系
试剂成分 | 试剂量 |
5X One Step Buffer | 6μL |
MSRV-F(10μM) | 0.6μL |
MSRV-R(10μM) | 0.6μL |
MSRV-P(10μM) | 0.3μL |
DNA Template | 1-100ng |
ddH2O(RNase-Free) | Up to 30μL |
将配制好的反应管离心,置于Roche 480II仪器中,设置PCR反应程序,程序如下:55℃15min,95℃预变性30s;95℃变性10s,60℃退火30s,共40个循环。
结果如图1和图2所示,出现“S”型曲线的为阳性,无“S”型曲线的则为阴性,两个实际样本和阳性质控品(核苷酸序列如SEQ ID No.1所示)都出现了“S”型曲线,而阴性对照未出现扩增。说明本发明的试剂盒对加州鲈鱼弹状病毒的检测结果是准确的。
实施例2
试剂盒的引物特异性实验。
为检测本试剂盒的引物特异性,采用实施例1中RT-PCR扩增检测方法,分别对虾白斑综合症病毒(WSSV)、传染性皮下及造血器官坏死病毒(IHHNV)、虾肝肠胞虫(EHP)、对虾桃拉综合症病毒(TSV)和虾血细胞虹彩病毒(SHIV)进行检测,分析本引物组和探针对MSRV病毒和其他水产常见病毒的检测情况。
检测结果如图3所示,检测结果表明,仅病毒MSRV样品出现“S”型扩增曲线,阴性对照(超纯水)及WSSV、IHHNV、EHP、TSV、SHIV病毒样品均未出现扩增。上述实验结果说明,加州鲈鱼弹状病毒检测引物组和探针能特异性扩增检测出病毒MSRV中的靶序列,而不与其它病毒核酸发生交叉反应。说明本发明的引物组和探针特异性好,未出现假阳性。
实施例3
灵敏度实验。
提取阳性标准品(PUC57-MSRV质粒,安徽通用合成),通过NanoDrop One对阳性质粒进行定量,并将其分别稀释到106、105、104、103、102、101浓度。采用实施例1中的检测方法,对稀释后的各浓度阳性标准品进行扩增检测。
检测结果如图4所示,从左到右的曲线依次为106、105、104、103、102、101的阳性标准品的扩增结果,从中可以看出本试剂盒最低可检测10拷贝的病毒核酸分子,精确性高,表明本发明的检测试剂盒和检测方法对病毒MSRV的诊断具有高度的灵敏性。
序列表
<110> 杭州奥盛仪器有限公司
浙江科技学院
浙江省水产技术推广总站
<120> 一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1527
<212> DNA
<213> 加州鲈鱼弹状病毒(Micropterus salmoides Rhabdoviridae)
<400> 1
atgaaattga tcattgcacc tacgctggtc tcccaggcga ttggctaccc gctgtttgtt 60
ccgataagac ttcaaggatg gcatgatgtt aaactagata ctctgaggtg tcccagttat 120
gcatctgagc tgaataagga ggccgcttgg ccacagattg gattgagaca tctcgctgcg 180
aaagatcatt atgaagtaaa agggacaatc tgtcataaga ccacctgggt gaaaacttgt 240
gacctcagat ggtatggacc taaatatatc acgaccaaga tttcgtacac acctataacc 300
ggattagaat gtcaacaggc aatagttaaa gcatctaaag atgagctaga gacaccatac 360
atgccagaag ataactgcga ttgggctaca atcagcgata atgaaaagac atttatcacc 420
gtccaaaaga gcaacatctt catggacccg tacaacatgg tctatgtaag cacagtctta 480
aaaggaggaa aatgtgcaag taccgtttgc ccactcgaaa tgcatggagg aatttggata 540
cccagtgagg cacccaggga gagttgccaa ctgggcagca gcatcaccag ccacatcaat 600
cccaacaacg catccaggtt aatatcagag gaaagttatt tggtcacaga gtatcataga 660
caactgccgt tcttgggagc ttgtaggatg tcaatgtgcg gagaggtggg aatgaggttt 720
aagtccggag aatggtacaa aattgagtca agcgacggac gggtgctgtc ctttctcagt 780
agtgttccaa tgtgtgatgg agagttgact gtctccatcc atgacggctc agctacgtat 840
cacaaattga gccaggaaat ccttgatctg tccgcacaaa tcgcctgcat atccgaatta 900
cgaagagccc gagagaaaaa tgcagttagc aattacctct tgagtttttt aacaccaaat 960
catggagggt tcgggacggc ataccgggtg ctcaacggac agttgcaatc ttctaaggcc 1020
acttatgtga gagtgaaact cggtgctctc tccaccgcga caaattgggg acaactagat 1080
gacggcagtg catactcttc ggaagatgtc actggcaaga tagttgatgg accgctattc 1140
aatgggaatc ggatggacaa tgggactctt agggtcgttc agaatgcaat attgggccag 1200
acactagaag atgaagattt gtatgaacac tcagcaaaag agattctcca tccgcatctg 1260
acagtcctga gtagcaatga atcagacgtg ctgtccgcat tccgtcccgt aggtgcccaa 1320
ggcgatatca tacatgcggt gggtgagtgg gtgggaactg gagtgagcgg atttatccat 1380
accattgtct atttggtgat cctctgtggc atcattcttc tcctttacag atgcttgcca 1440
tatcttttga aaaaacgaaa atcacaatcg acgagtcaga cgactcctca aatgatccct 1500
ttgcagcagt atcaatttgt tccctaa 1527
<210> 2
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
agacaccata catgccagaa g 21
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gttgtacggg tccatgaaga t 21
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgcgattggg ctacaatcag cgat 24
Claims (1)
1.一种非疾病诊断为目的的加州鲈鱼弹状病毒的检测方法,其特征在于,包括以下步骤:
(1)提取待测样品病毒RNA,将待测样品病毒RNA逆转录成cDNA;
(2)以步骤(1)中的cDNA作为扩增模板,使用引物对和探针进行特异性RT-PCR扩增;
所述引物对包括正向引物和反向引物,正向引物的核苷酸序列如SEQ ID No.2所示,反向引物的核苷酸序列如SEQ ID No.3所示;所述探针的核苷酸序列如SEQ ID No.4所示,所述探针的5’端标记荧光染料FAM,3’端标记淬灭基团BHQ1;
(3)若能够扩增得到信号,说明待检测样品中含有加州鲈鱼弹状病毒;
步骤(2)中RT-PCR反应体系为:5×One Step Buffer 6μL,正向引物和反向引物各为6pmol,探针为3pmol,DNA模板为1-100ng,无菌无核酸酶水补至30μL;
步骤(2)中RT-PCR扩增反应条件为55℃15min,95℃预变性30s;95℃变性10s,60℃退火30s,共40个循环。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210276386.3A CN114592091B (zh) | 2022-03-18 | 2022-03-18 | 一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210276386.3A CN114592091B (zh) | 2022-03-18 | 2022-03-18 | 一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN114592091A CN114592091A (zh) | 2022-06-07 |
CN114592091B true CN114592091B (zh) | 2023-11-03 |
Family
ID=81811139
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210276386.3A Active CN114592091B (zh) | 2022-03-18 | 2022-03-18 | 一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114592091B (zh) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111187862A (zh) * | 2020-03-11 | 2020-05-22 | 浙江省淡水水产研究所 | 一种基于重组酶的大口黑鲈弹状病毒恒温扩增检测试剂盒 |
CN113637802A (zh) * | 2021-08-24 | 2021-11-12 | 中国水产科学研究院珠江水产研究所 | 一种利用数字pcr技术检测鲈弹状病毒的试剂盒及方法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007075988A2 (en) * | 2005-12-21 | 2007-07-05 | Advanced Bionutrition Corporation | Non-invasive detection of fish viruses by real-time pcr |
-
2022
- 2022-03-18 CN CN202210276386.3A patent/CN114592091B/zh active Active
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN111187862A (zh) * | 2020-03-11 | 2020-05-22 | 浙江省淡水水产研究所 | 一种基于重组酶的大口黑鲈弹状病毒恒温扩增检测试剂盒 |
CN113637802A (zh) * | 2021-08-24 | 2021-11-12 | 中国水产科学研究院珠江水产研究所 | 一种利用数字pcr技术检测鲈弹状病毒的试剂盒及方法 |
Non-Patent Citations (2)
Title |
---|
Sun-Jian Lyu等.Isolation and characterization of a novel strain (YH01) of Micropterus salmoides rhabdovirus and expression of its glycoprotein by the baculovirus expression system.J Zhejiang Univ Sci B..2019,第20卷(第9期),第3.2节和图1. * |
Xiaozhe Fu等.The biological features and genetic diversity of novel fish rhabdovirus isolates in China.Arch Virol. .2017,第162卷(第9期),2829-2834. * |
Also Published As
Publication number | Publication date |
---|---|
CN114592091A (zh) | 2022-06-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111187862B (zh) | 一种基于重组酶的大口黑鲈弹状病毒恒温扩增检测试剂盒 | |
CN111534635B (zh) | 一体化分子诊断系统及其在追溯β冠状病毒动物来源的应用 | |
CN111118218B (zh) | 对虾虹彩病毒的CRISPR-Cas12a蛋白酶等温检测引物组、试剂盒及其检测方法 | |
CN112176105B (zh) | 病毒bvdv、brv和bcv一管多重荧光pcr检测的专用引物及其应用 | |
CN111394515B (zh) | 一种用于检测犬细小病毒的lamp引物组、荧光可视化快速试剂盒及方法 | |
CN106947834B (zh) | 一种检测六种鸭易感病毒的多重pcr方法 | |
Gui et al. | Quick detection of Carassius auratus herpesvirus (CaHV) by recombinase-aid amplification lateral flow dipstick (RAA-LFD) method | |
CN113943831A (zh) | 可同时诊断猪腹泻病三种高发病原的多重荧光定量引物和探针组合及其应用 | |
CN114592091B (zh) | 一种用于加州鲈鱼弹状病毒检测的试剂盒及其检测方法 | |
CN110387440B (zh) | 一种用于鲑鳟鱼病毒多重检测的试剂及其应用 | |
CN112941240B (zh) | 检测鹅星状病毒和鹅嵌杯病毒的引物对、试剂盒及方法 | |
CN113430274B (zh) | 一种检测肝肠胞虫的rpa引物、探针、试剂盒及方法 | |
CN115094164A (zh) | 不同基因缺失型ASFV的多重qPCR试剂盒及检测方法 | |
CN110592269A (zh) | 草鱼出血病2型病毒(gcrv-2)的raa恒温荧光检测方法及试剂 | |
CN107604101A (zh) | 一种鸽新型腺病毒实时荧光定量pcr检测试剂盒 | |
CN114381551A (zh) | 一种用于检测大口黑鲈虹彩病毒的实时荧光raa引物、探针及试剂盒 | |
CN109897918B (zh) | 鲤浮肿病毒和锦鲤疱疹病毒双重实时荧光定量检测方法 | |
CN109182597B (zh) | 一种同时检测禽腺病毒i、ii、iii群多重荧光定量pcr试剂盒及其检测方法 | |
CN106521038A (zh) | 一种高灵敏度的bhv‑2实时荧光定量pcr检测方法及试剂盒 | |
CN113151586A (zh) | 一种用于检测、鉴别猪伪狂犬病毒ⅰ型和ⅱ型的引物组合、试剂盒及方法 | |
CN108148917B (zh) | 一种检测高致病性副溶血弧菌的lamp-lfd的引物和探针及其应用 | |
CN115961051A (zh) | 日本血吸虫w染色体特异基因的筛选及其在尾蚴性别鉴定中的应用 | |
CN110616279A (zh) | 一种同步定量检测3种重要虾类病原的试剂盒 | |
CN110894551A (zh) | 草鱼出血病i型病毒(gcrv-i)的raa恒温荧光检测方法及试剂 | |
CN102634532A (zh) | 传染性造血器官坏死病毒分子标准样品的制备方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |