CN102851403B - Cyprinidherpesvirus 2 (CyHV-2) nested PCR (polymerase chain reaction) detection kit - Google Patents
Cyprinidherpesvirus 2 (CyHV-2) nested PCR (polymerase chain reaction) detection kit Download PDFInfo
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Abstract
The invention discloses a Cyprinidherpesvirus 2 (CyHV-2) nested PCR (polymerase chain reaction) detection kit which is characterized in that six 1.5mL centrifuge tubes containing reagents are arranged on an inner pad (2) of a box body (1). The kit comprises a 10* reaction buffer, primers P1 and primers P2, wherein the 10* reaction buffer contains 100mM of Tris-HCL, 500mM of KCl, 15mM of MgCl2, 2mM of dNTPs (deoxyribonucleotide triphosphates) (3), and 1U/mu L Taq enzyme (4); the primers P1 contain 10mu M of CyHV-2 P1F and 10mu M of CyHV-2 P1R (5); and the primers P2 contain 10mu M of CyHV-2 P2F and 10mu M of CyHV-2 P2R (6), pure water (7) and 10mu g/mL positive DNA (deoxyribonucleic acid) (8). The sequences of the outer primers P1 are as follows: CyHV-2 P1F: TGAAATGTCAAAAGTGGATGG, and CyHV-2 P1R: TATTCCCAGACAGCCTTCAAA; and the sequences of the inner primers P2 are as follows: CyHV-2 P2F: GAACACCGCTGCTCATCATC, and CyHV-2 P2R: ACTCTTCGCAAGTCCTCACC. Two amplification processes are carried out by the inner and outer primers to finally obtain the DNA product of 357bp. The invention is simple to operate, is convenient and quick, and has the advantages of low detection limit, high sensitivity and accurate and reliable detection result.
Description
Technical field
The present invention relates to aquatic animal cause of disease detection field, (Cyprinidherpesvirus 2 more specifically to relate to a kind of carp herpes virus type 2, CyHV-2) gene detecting kit, be applicable to carp blister sore viral disease 2 types in aquaculture diagnosis, import and export the rapid detection of carp blister sore viral disease 2 types in inspection and quarantine.
Background technology
Carp herpes virus type 2 (CyHV-2) is downright bad sick (the herpesviral haematopoietic necrosis of blister sore toxicity hemocytopoietic organ that causes goldfish mortality, HVHN) cause of disease, therefore also referred to as goldfish hematopoietic necrosis virus (goldfish haematopoietic necrosis virus, GFHNV).According to the systematic naming method rule of the world viral phylogenetic systematics council, definite designation be carp herpes virus type 2 (Cyprinid herpesvirus 2, CyHV-2).This virus is autumn in 1992 and spring in 1993 because causing the goldfish mortality of western Japan cultivation to be found first, and its lethality rate is up to 100%.This disease is subsequently in the U.S., TaiWan, China, and Australia and Britain are broken out in succession, to goldfish, cultivate and have caused huge financial loss.In recent years, there is extensive death in the crucian of the main culture zone of China crucian (hybridized prussian carp), and mortality ratio reaches more than 80%, through electron microscopic observation and Molecular Detection, determines that cause of disease is CyHV-2.This disease spread scope is wide, infectivity is strong, rapid onset, lethality rate are high, and Yi Gei China crucian cultivation industry has caused huge financial loss, becomes one of the most serious virus disease of harm in China's aquaculture.
Cell cultures isolation technique is the effective ways of viral diagnosis, the prefered method that the fishes virus that normally OIE (OIE) is recommended detects.But existing research data shows, CyHV-2 is difficult to propagation in existing fish cell system.Fat carp cell (fathead minnow cells, FHM), carp epithelioma cell (epithelioma papillosum cyprini, EPC), eel nephrocyte (eel kidney, EK-1), salmon embryonic cell (chinook salmon embryo, CHSE-214), rainbow trout gonadal cell (rainbow trout gonad, RTG-2) and tilapia gonad cell (tilapia ovary, TO-2) all insensitive to CyHV-2.Only (koi fin 1, KF-1) can produce distinctive cytopathic effect (CPE), but it's a pity bright and beautiful carp fin cell, and virus is uploaded to after the 3rd generation at KF-1 cell, and CPE disappears.The visible current stage, cell cultures is not the effective ways of CyHV-2 diagnosis, and the technology detecting based on virogene just becomes the most effective detection method of CyHV-2.Polymerase chain reaction (Polymerase Chain Reaction, PCR) amplification technique, as the most basic gene amplification, is widely used in molecular biological every field now, in virus, identifies that field is also widely used.Yet its amplification is limited to the template content of detected object, when template amount is seldom time, usually obtain negative findings.In science of fish disease field, the virus quantity in ill fish body is conventionally very low, uses traditional conventional PCR detection technique conventionally because false negative result appears in its insufficient sensitivity, cannot meet aquiculture disease diagnosis and port quarantine requirements of one's work.Nest-type PRC amplification technique is a kind of new technology being based upon on conventional round pcr, and its design two cover PCR primers (nested primer) carry out two-wheeled pcr amplification reaction to template.In first round amplification, outer primer is in order to produce amplified production, usings this amplified production as template, with inner primer, to carry out second and take turns amplification.Because nest-type PRC reaction has twice PCR amplification, thereby reduced the possibility (all complementary template is seldom because with two cover primers) of a plurality of target sites that increase thereby increased the susceptibility detecting, there are again two pairs of PCR primers and the pairing that detects template, increased the reliability detecting simultaneously.Have no at present the report of the nest-type PRC test kit of CyHV-2.
Summary of the invention
For the deficiencies in the prior art, the object of the invention is to be to provide a kind of nest type PCR detection reagent of carp herpes virus type 2, the present invention is simple to operate, convenient and swift, and detectability is low, highly sensitive, and detected result is accurately and reliably.
The present invention using extracting from the viral DNA of ill fish tissue as pcr amplification template, with two pairs of nested type primer pair template DNAs, increase, can complete whole PCR within a short period of time detects, not only greatly improved detection sensitivity, and shortened detection time, and result accurately and reliably, it is a kind of effective carp herpes virus type 2 molecular biology for detection.
In order to realize above-mentioned object, the present invention by the following technical solutions:
A kind of nest type PCR detection reagent of carp herpes virus type 2, comprise box body, in box inner liner, place 6 1.5mL centrifuge tubes that fill reagent, comprise 10 * reaction buffer, Taq enzyme (1U/ μ L), primer P1 (containing each 10 μ M of CyHV-2P1F, CyHV-2P1R), primer P2 (containing each 10 μ M of CyHV-2P2F, CyHV-2P2R), pure water (molecular biology grade), positive DNA (10 μ g/mL).
The formula of 10 described * reaction buffer is: Tris-HCl 100mM, KCl 500mM, MgCl215mM, dNTPs2mM.
Described Tris-HCl is Tri(Hydroxymethyl) Amino Methane Hydrochloride; KCl is Repone K; MgCl
2for magnesium chloride; DNTPs is the equal-volume mixed solution of four kinds of required deoxynucleotides of synthetic DNA.
Above-mentioned various liquid reagent is contained in respectively in 1.5mL centrifuge tube, is placed in box.
Described primer nucleotides sequence is classified as:
CyHV-2P1F is: TGAAATGTCAAAAGTGGATGG
CyHV-2P1R is: TATTCCCAGACAGCCTTCAAA
CyHV-2P2F is: GAACACCGCTGCTCATCATC
CyHV-2P2R is: ACTCTTCGCAAGTCCTCACC
A nest type PCR detection reagent for carp herpes virus type 2, its detecting step is:
1) detect the extraction of sample DNA before detecting: get appropriate dying ill Yu Gill silk, spleen, renal tissue, use glass homogenizer homogenate, 5000rpm in 4 ℃ centrifugal 10 minutes, get DNA extraction reagent extraction DNA for appropriate homogenate supernatant liquor.
2) pcr amplification of outer primer P1: preparation PCR reaction system 20 μ L:10 * PCR reaction buffer 2 μ L, Taq enzyme (1U/ μ L) 1 μ L, outer primer P10.5 μ L, DNA profiling 4 μ L, pure water complements to 20 μ L.Get positive control dna template simultaneously and do positive control, get pure water and do negative control.PCR reaction conditions is: 94 ℃ of denaturations 5 minutes, and 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are extended 40 seconds, rear 72 ℃ of 30 circulations 5 minutes.
3) pcr amplification of inner primer P2: get the amplified production of 1 μ L outer primer P1 as template, the inner primer P2 of take carries out pcr amplification as primer, and other reacted constituents and reaction conditions are with amplification for the first time.
4) amplified production detects and analyzes: 1 ~ 2% (W/V) agarose gel electrophoresis testing goal band imaging analysis.
5) result is judged:
A. in the same size when the amplified band of detection sample and positive control, during for 357bp, result is positive;
B. when detecting amplified band not of uniform size cause of sample without band or stripe size and positive control, while being not 357bp, result is negative;
C. when positive control is during without band, show related reagent degraded in test kit, test kit lost efficacy;
D. when band appears in negative control, have reagent contamination in operating process, detected result is invalid.
The invention has the beneficial effects as follows:
The present invention detects the nest-type PRC test kit of carp herpes virus type 2, and a kind of molecular detection technology that detects highly pathogenic fishes virus is provided, and especially can detect quick, easy, accurately the test kit of cause of disease, can in 8 hours, complete detection.
The invention provides the molecular detection kit of a kind of PCR of dependence, overcome that the viral diagnosis methods such as cellular segregation cultivation, the observation of Electronic Speculum ultrathin section(ing), ELISA detection are loaded down with trivial details, efficiency is low, poor reliability, deficiency that cost is high.This test kit can detect cause of disease quick, sensitive, exactly, and can directly from ill fish tissue, virus causing disease be detected.Greatly simplified trace routine, improved detection level, the formulation for disease control strategy simultaneously provides technical basis.
By the specific detection to carp herpes virus type 2, through outer primer CyHV-2P1F and CyHV-2P1R, and inner primer CyHV-2P2F and CyHV-2P2R carry out two-wheeled pcr amplification to target viral, obtain the single PCR product of 357bp, detection target viral that can be special.But for 4 kinds of DNA virus such as Koi herpesvirus (KHV), giant salamander irido virus (GSIV), channel catfish virus (CCV), eel simplexviruss (HVA), all without amplified production.
By the detection of sensitivity, to clone, there is the T carrier of goal gene to carry out conventional PCR and nest-type PRC detection comparison, find that the lowest detection of conventional pcr amplification is limited to 10
3individual copy, and nest-type PRC lowest detection is limited to 10 copies.
Accompanying drawing explanation
Fig. 1 is a kind of schematic diagram of nest type PCR detection reagent of carp herpes virus type 2.
Wherein 1 is box body, and 2 is liner, and 3 is 10 * reaction buffer, and 4 is Taq enzyme, and 5 is primer P1, and 6 is primer P2, and 7 is pure water, 8 positive DNA.
Fig. 2 is the CyHV-2 detected result schematic diagram of the ill fish of inventive embodiments.
Wherein M is Takara DL1000DNA Marker, and 1 ~ 5 for detecting sample, 6 positive contrasts, 7 negative contrasts.
Embodiment
Below in conjunction with embodiment, principle of the present invention and feature are described, illustrated embodiment, only for explaining the present invention, is not intended to limit scope of the present invention.
Embodiment 1:
A kind of nest type PCR detection reagent of carp herpes virus type 2, comprise box body (1), 6 1.5mL centrifuge tubes that fill reagent of the upper placement of box inner liner (2), comprise 10 * reaction buffer (3), Taq enzyme (1U/ μ L) (4), primer P1 (comprising each 10 μ M of CyHV-2P1F, CyHV-2P1R) (5), primer P2 (comprising each 10 μ M of CyHV-2P2F, CyHV-2P2R) (6), pure water (molecular biology grade) (7), positive DNA (10 μ g/mL) (8).
The formula of 10 described * reaction buffer is: Tris-HCl 100mM, KCl 500mM, MgCl
215mM, dNTPs2mM.
Described Tris-HCl is Tri(Hydroxymethyl) Amino Methane Hydrochloride; KCl is Repone K; MgCl
2for magnesium chloride; DNTPs is the equal-volume mixed solution of four kinds of required deoxynucleotides of synthetic DNA.
Above-mentioned various liquid reagent is contained in respectively in 1.5mL centrifuge tube, is placed in box.
Described primer nucleotides sequence is classified as:
CyHV-2P1F is: TGAAATGTCAAAAGTGGATGG
CyHV-2P1R is: TATTCCCAGACAGCCTTCAAA
CyHV-2P2F is: GAACACCGCTGCTCATCATC
CyHV-2P2R is: ACTCTTCGCAAGTCCTCACC
Embodiment 2:
A kind of nest type PCR detection reagent of carp herpes virus type 2, comprise box body 1, in box inner liner 2, place 6 1.5mL centrifuge tubes that fill reagent, comprise 10 * reaction buffer 3, Taq enzyme (1U/ μ L) 4, primer P1 (comprising each 10 μ M of CyHV-2P1F, CyHV-2P1R) 5, primer P2 (comprising each 10 μ M of CyHV-2P2F, CyHV-2P2R) 6, pure water (molecular biology grade) 7, positive DNA (10 μ g/mL) 8.
The formula of 10 described * reaction buffer is: Tris-HCl 100mM, KCl 500mM, MgCl215mM, dNTPs2mM.
Described Tris-HCl is Tri(Hydroxymethyl) Amino Methane Hydrochloride; KCl is Repone K; MgCl
2for magnesium chloride; DNTPs is the equal-volume mixed solution of four kinds of required deoxynucleotides of synthetic DNA.
The using method of described test kit, comprises the following steps:
1) detect the extraction of sample DNA before detecting: get appropriate dying ill Yu Gill silk, spleen, renal tissue, use glass homogenizer homogenate, 5000rpm in 4 ℃ centrifugal 10 minutes, get DNA extraction reagent extraction DNA for appropriate homogenate supernatant liquor.
2) pcr amplification of outer primer P1: preparation PCR reaction system 20 μ L:10 * PCR reaction buffer 2 μ L, Taq enzyme (1U/ μ L) 1 μ L, outer primer P10.5 μ L, DNA profiling 4 μ L, pure water complements to 20 μ L.Get positive control dna template simultaneously and do positive control, get pure water and do negative control.PCR reaction conditions is: 94 ℃ of denaturations 5 minutes, and 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ are extended 40 seconds, rear 72 ℃ of 30 circulations 5 minutes.
3) pcr amplification of inner primer P2: get the amplified production of 1 μ L outer primer P1 as template, the inner primer P2 of take carries out pcr amplification as primer, and other reacted constituents and reaction conditions are with amplification for the first time.
4) amplified production detects and analyzes: 1 ~ 2% (W/V) agarose gel electrophoresis testing goal band imaging analysis.
5) result is judged:
E. in the same size when the amplified band of detection sample and positive control, during for 357bp, result is positive;
F. when detecting amplified band not of uniform size cause of sample without band or stripe size and positive control, while being not 357bp, result is negative;
G. when positive control is during without band, show related reagent degraded in test kit, test kit lost efficacy;
H. when band appears in negative control, have reagent contamination in operating process, detected result is invalid.
Embodiment 3:
Detection to the ill fish tissue samples of 5 natural infections:
Randomly draw the crucian that suffers from typical illness, get Shi Liang Gill and be organized in glass homogenizer homogenate, get homogenate supernatant liquor 0.25mL and extract viral DNA template with DNAzol, with the reagent in test kit, carry out nest-type PRC detection.1% (W/V) agarose gel electrophoresis and imaging system analysis for detected result.Result shows (accompanying drawing 2), and 5 crucian samples that detect are the CyHV-2 positive, and this result and Electronic Speculum ultrathin section(ing) observations are in full accord.
The foregoing is only preferred embodiment of the present invention, in order to limit this enforcement, within the spirit and principles in the present invention not all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
SEQUENCE LISTING
<110> Changjiang Aquatic Products Inst., Chinese Academy of Aquatic Products Sciences
The nest type PCR detection reagent of a <120> carp herpes virus type 2
The nest type PCR detection reagent of a <130> carp herpes virus type 2
<160> 4
<170> PatentIn version 3.1
<210> 1
<211> 21
<212> DNA
<213> synthetic
<400> 1
tgaaatgtca aaagtggatg g 21
<210> 2
<211> 21
<212> DNA
<213> synthetic
<400> 2
tattcccaga cagccttcaa a 21
<210> 3
<211> 20
<212> DNA
<213> synthetic
<400> 3
gaacaccgct gctcatcatc 20
<210> 4
<211> 20
<212> DNA
<213> synthetic
<400> 4
actcttcgca agtcctcacc 20
Claims (1)
1. the nest type PCR detection reagent of a carp herpes virus type 2, it is characterized in that: 6 1.5mL centrifuge tubes that fill reagent of the upper placement of box body (1) inner liner (2), comprise 10 * reaction buffer: containing Tris-HCl100mM, KCl500mM, MgCl215mM, dNTPs2mM (3), Taq enzyme 1U/ μ L (4), primer P1: containing each 10 μ M (5) of CyHV-2P1F, CyHV-2P1R, primer P2: containing each 10 μ M (6) of CyHV-2P2F, CyHV-2P2R, pure water (7), positive DNA10 μ g/mL (8);
Above-mentioned various liquid reagent is contained in respectively in 1.5mL centrifuge tube, is placed in box;
CyHV-2P1F is: TGAAATGTCAAAAGTGGATGG
CyHV-2P1R is: TATTCCCAGACAGCCTTCAAA
CyHV-2P2F is: GAACACCGCTGCTCATCATC
CyHV-2P2R is: ACTCTTCGCAAGTCCTCACC.
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| CN106857338A (en) * | 2017-04-17 | 2017-06-20 | 中国水产科学研究院长江水产研究所 | A kind of crucian carp Hematopoietic Necrosis disease Synthetical prevention method |
| CN108070678A (en) * | 2018-02-01 | 2018-05-25 | 上海海洋大学 | Constant temperature detects the RPA kits of II type carp herpesvirals and its primer special and probe in real time |
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| CN103088163B (en) * | 2013-01-16 | 2015-07-01 | 深圳出入境检验检疫局动植物检验检疫技术中心 | Method and primer for detecting necrosis virus of goldfish hematopoietic organ by loop-mediated isothermal amplification |
| CN103045764A (en) * | 2013-01-18 | 2013-04-17 | 中国水产科学研究院长江水产研究所 | CyHV-2 (Cyprinid herpesvirus II) LAMP (loop-mediated isothermal amplification) detection kit and detection method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106857338A (en) * | 2017-04-17 | 2017-06-20 | 中国水产科学研究院长江水产研究所 | A kind of crucian carp Hematopoietic Necrosis disease Synthetical prevention method |
| CN108070678A (en) * | 2018-02-01 | 2018-05-25 | 上海海洋大学 | Constant temperature detects the RPA kits of II type carp herpesvirals and its primer special and probe in real time |
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