CN104878124A - Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus) - Google Patents
Multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus) Download PDFInfo
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Abstract
The invention discloses a multiple PCR (Polymerase Chain Reaction) detection kit for duck hepatitis A virus 1 and 3 as well as MDPV (Muscovy Duck Parvovirus). The detection kit contains three pairs of specific primers. Experimental results show the multiple PCR detection kit for duck hepatitis A virus 1 and 3 as well as MDPV can be applied to detecting the duck hepatitis A virus 1 and 3 and the MDPV. The multiple PCR detection kit has the advantages of being convenient to operate, high in sensitivity, strong in specificity and the like; furthermore, the multiple PCR detection kit can be clinically used for distinguishing and detecting the duck hepatitis A virus 1 and 3 and the MDPV, the time cost can be saved, and the pollution can be reduced.
Description
Technical field
The invention belongs to PCR detection technique field, particularly relate to a kind of duck hepatitis A virus (HAV) 1,3 type and Muscovy duck parvovirus multiple PCR detection kit.
Background technology
Duck hepatitis A virus (HAV) can to cause within 3 week age duckling liver enlargement and hemorrhage, to the lethality rate of duckling up to more than 90%, is that one of cause of disease the most serious of duck industry development is supported in harm.Duck hepatitis A virus (HAV) can be divided into duck hepatitis A virus (HAV) 1 type (DHAV-1), duck hepatitis A virus (HAV) 2 type (DHAV-2) and duck hepatitis A virus (HAV) 3 type (DHAV-3) according to its serotype, respectively corresponding traditional serum I type, the novel and Korean Utility duck hepatitis virus in Taiwan.At present, the duck hepatitis A virus (HAV) that China is popular is mainly DHAV-1 and DHAV-3.Muscovy duck parvovirus (MDPV) is duckling within 3 week age of infringement mainly, infect and can to play duckling after duckling and breathe, suffer from diarrhoea and Pancreatic Necrosis and hemorrhage, can reach more than 40% to the lethality rate of duckling is also one of transmissible disease the most serious of developing of restriction China kind duck animal husbandry.Because DHAV-1, DHAV-3 and MDPV are the most susceptible of duckling and the very high virus of lethality rate, and the duck viral hepatitis clinical symptom that DHAV-1 and DHAV-3 causes and pathological change closely similar so that usually can not diagnose in time with effective prevention and control and give cultivation industrial belt come tremendous economic loss.
At present, virus purification, serological test, agar diffusion test and enzyme linked immunosorbent assay etc. are mainly contained to the traditional detection method of these viruses, but the limitation such as length consuming time, susceptibility are poor, difficult stdn that these methods exist, especially, when this two-strain polyinfection, the difficulty of clinical diagnosis is more the increase in.
Round pcr achieves the detection by quantitative to template, and have sensitivity, special, accurately and reliably, can the features such as multiple reaction be realized.In actual applications, when sample size is very large, just there is certain inferior position at cost with in the time in substance PCR, carries out rapid detection in batches in the urgent need to a kind of high-throughput, low cost, high efficiency method.Multiplex PCR adopts multipair primer amplification to detect multiple template, overcomes the deficiency of substance PCR.But set up multiple PCR method more more complex than substance, it is higher to the requirement of reagent and primer, need to ensure between different primers without interference mutually simultaneously.
Summary of the invention
The technical problem to be solved in the present invention is to provide that a kind of susceptibility is high, high specificity, the hepatitis A virus (HAV) 1 of duck fast type easy and simple to handle, 3 types and Muscovy duck parvovirus multiple PCR detection kit.
For solving the problems of the technologies described above, the present invention is by the following technical solutions: duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus multiple PCR detection primer group, comprise three pairs of Auele Specific Primers, be primer 1 and 2 (detecting DHAV-1), primer 3 and 4 (detecting DHAV-3), primer 5 and 6 (detecting MDPV) respectively, they have the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6 respectively.
The mol ratio of primer 1, primer 2, primer 3, primer 4, primer 5, primer 6 is 0.5-1: 0.5-1: 1: 1: 1: 1.
The mol ratio of primer 1, primer 2, primer 3, primer 4, primer 5, primer 6 is 1: 1: 1: 1: 1: 1.
The non-diagnostic application in pcr amplification of above-mentioned duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus multiple PCR detection primer group, the annealing temperature of pcr amplification is 50.0 DEG C-60.1 DEG C.
The annealing temperature of pcr amplification is 50.0 DEG C.
Duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus multiple PCR detection kit, comprise primer sets, PCR damping fluid and water;
Primer sets comprises three pairs of Auele Specific Primers, primer 1 and 2, primer 3 and 4, primer 5 and 6 respectively, they have the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6 respectively, its concentration is 25 μm of ol/mL, and the final concentration in PCR reaction system is respectively 0.1 μm of ol/mL-0.5 μm of ol/mL, 0.1 μm of ol/mL-0.5 μm of ol/mL, 0.5 μm of ol/mL-1.0 μm of ol/mL;
PCR damping fluid is 2 × Taq PCR Mix.
Primer 1 and 2, primer 3 and 4, the final concentration of primer 5 and 6 in PCR reaction system are respectively 0.5 μm of ol/mL, 0.3 μm of ol/mL, 0.7 μm of ol/mL.
The non-diagnostic application in pcr amplification of above-mentioned duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus multiple PCR detection kit, the annealing temperature of pcr amplification is 50.0 DEG C-60.1 DEG C.
The annealing temperature of pcr amplification is 50.0 DEG C.
For lacking the technology effectively reliably of duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus being carried out to diagnosis and detection at present, contriver manages the advantages such as many inspection, high-throughput, low cost, high-level efficiency in conjunction with multiplex PCR one, the research and design three pairs of Auele Specific Primers, establish the multi-PCR detection method differentiated and detect duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus simultaneously accordingly, and prepare corresponding detection kit.Experiment proves, application the present invention can detect DHAV-1, DHAV-3 and MDPV, and can differentiate to detect DHAV-1, DHAV-3 simultaneously, has the advantages such as easy and simple to handle, susceptibility is high, high specificity, both can time-saving cost, again can decreasing pollution.
Accompanying drawing explanation
Fig. 1 is the specific test result electrophorogram of multiplex PCR, in figure, and M:100bp DNA ladder; 1:DHAV-1cDNA+DHAV-3cDNA+MDPV cDNA (DHAV-1, DHAV-3cDNA 1 μ L, MDPV cDNA 2 μ L); The cDNA of 2:DHAV-1; The cDNA of 3:DHAV-3; The cDNA of 4:MDPV; The cDNA of 5:H5AIV; The cDNA of 6:H9AIV; 7: the cDNA of muscovy duck reovirus; 8: duck circovirus DNA; 9: the cDNA of duck paramyxoviru; 10: the cDNA of duck hepatitis virus; 11: the DNA of duck plague virus; 12: negative control (ddH
2o).
Fig. 2 is the sensitivity test result electrophorogram of multiplex PCR, in figure, and M:100bp DNA ladder; 1:52ng/ μ LDHAV-1,79ng/ μ L DHAV-3,58ng/ μ L MDPV cDNA; 2:5.2ng/ μ L DHAV-1,7.9ng/ μ L DHAV-3,5.8ng/ μ L MDPV cDNA; 3:520pg/ μ L DHAV-1,790pg/ μ L DHAV-3,580pg/ μ L MDPV cDNA; 4:52pg/ μ L DHAV-1,79pg/ μ L DHAV-3,58pg/ μ L MDPV cDNA; 5:5.2pg/ μ L DHAV-1,7.9pg/ μ L DHAV-3,5.8pg/ μ L MDPV cDNA; 6:0.52pg/ μ L DHAV-1,0.79pg/ μ L DHAV-3,0.58pg/ μ L MDPV cDNA.
Fig. 3 is the partial clinical sample detection result electrophorogram applying multiplex PCR of the present invention, and in figure, M is 100bp DNAladder; 1 is positive control (DHAV-1 cDNA+DHAV-3 cDNA+MDPV cDNA); 2-17 is the duck pathological material of disease that laboratory gathers.
Embodiment
The experimental technique used in following examples if no special instructions, is ordinary method; Material used, reagent etc., if no special instructions, all can obtain from commercial channels.Concrete material therefor and reagent as follows:
Duck hepatitis virus (DHAV-1) is documented in " foundation of duck I Hepatitis virus RT-LAMP visible detection method ", China's Preventive Veterinary Medicine report, 2012,34 (2): 112-115, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
Duck hepatitis virus (DHAV-3) is documented in " separation of New Type Duck Hepatitis Virus and preliminary evaluation ", Guangxi animal and veterinary, and 2003,19 (5): 198-199, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region.
Duck plague virus AV1221 is purchased from China Veterinery Drug Inspection Office;
Muscovy duck parvovirus is documented in " separation andpreconcentration of Guangxi Muscovy duck parvovirus ", Guangxi animal and veterinary, and 2002,18 (6): 5-7, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Duck circovirus is documented in " investigation of some areas of Guangxi duck circovirus infection conditions ", Chinese animal and veterinary, and 2010,37 (11): 156-158, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Duck paramyxoviru (duck Avian pneumo-encephalitis virus) is documented in " foundation of duck Avian pneumo-encephalitis virus and duck circovirus duplex PCR detection method ", China's Animal Quarantine, 2012,29 (5): 34-37, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
Muscovy duck reovirus is documented in " research of kind duck flower hepatopathy cause of disease ", Chinese animal doctor's science and technology, and 2003,5 (33): 7-8, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H5 subtype avian influenza virus is documented in " foundation of H5 subtype avian influenza virus RT-LAMP Visual retrieval technology ", agriculture in south journal, 2013,02:323-327, and the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
H9 subtype avian influenza virus is documented in " multiplex reverse polymerase chain reaction rapid detection differentiates the foundation of H9 subtype avian influenza virus method ", China Amphixenosis journal, 2006, (09): 858-860, the public can obtain from Veterinary Institute of Guangxi Zhuang Autonomous Region;
100bp Marker ladder, 2 × Taq PCR Mix (AS111-12), DNA/RNA extract test kit, Reverse Transcription all purchased from Beijing Quanshijin Biotechnology Co., Ltd.
The Design and synthesis of embodiment 1, primer
According to the conserved sequence of the VP0 gene of DHAV-1 3C gene, DHAV-3 and the NS gene of MDPV in GenBank, adopt DNAStar software to carry out Multiple Sequence Alignment, design primer at conserved regions primer 5.0.Primer is synthesized by Beijing Liuhe Huada Genomics Technology Co., Ltd.(table 1).
Table 1 primer information
Embodiment 2, multiplex PCR detect
One, the extraction of nucleic acid and the reverse transcription of RNA
Test kit specification sheets is extracted with reference to DNA/RNA, extract Muscovy duck parvovirus, duck circovirus, the DNA of duck plague virus, simultaneously to H5, H9, duck paramyxoviru, duck hepatitis virus, the RNA of muscovy duck reovirus carries out extracting, and by reverse transcription specification sheets, RNA reverse transcription is become cDNA, specific as follows: to adopt 10 μ L systems, 2 μ L 5 × reverse transcription buffer are added successively in EP pipe, 10mmol/L dNTP 1 μ L, 5U/ μ LAMV 0.5 μ L, 20U/ μ L RNA enzyme inhibitors 0.5 μ L, free primer 0.5 μ L, total serum IgE template 2 μ L to be checked, supplying volume with the aqua sterilisa without RNA enzyme is 10 μ L, wink is from rearmounted PCR instrument, 42 DEG C of 1h, 85 DEG C of 5min, the cDNA of preparation puts-30 DEG C and saves backup.
Two, the foundation of multiplexed PCR amplification system
Total reaction volume is 25 μ L, wherein 2 × PCR Master Mix 12.5 μ L, DHAV-1cDNA, DHAV-3cDNA and MDPV cDNA totally 4 μ L as hybrid template, primer DHAV-1-F, DHAV-1-R, DHAV-3-F, DHAV-3-R, MDPV-F, MDPV-R are appropriate, finally mend to 25 μ L with distilled water.Each primer concentration of multiplex PCR and each parameter (temperature, time) of PCR reaction are optimized, to filter out reaction pattern best in duplex PCR reaction system.
To DHAV-1 primer, DHAV-3 primer, MDPV primer, the consumptions such as template are optimized, finally determine that multi-PRC reaction system is 25 μ L:2 × PCR Master Mix (full formula gold in Beijing, AS111-12) 12.5 μ L, (primer concentration is 25 μm of ol/mL to each 0.5 μ L of DHAV-1 upstream and downstream primer, final concentration in reaction system is 0.5 μm of ol/mL), (primer concentration is 25 μm of ol/mL to each 0.3 μ L of DHAV-3 upstream and downstream primer, final concentration in reaction system is 0.3 μm of ol/mL), (primer concentration is 25 μm of ol/mL to each 0.7 μ L of MDPV upstream and downstream primer, final concentration in reaction system is 0.7 μm of ol/mL), template is totally 4 μ L, add water to 25 μ L.
According to above-mentioned reaction system, template is the cDNA mixture of duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus, annealing temperature is increased progressively successively by 50.0 DEG C-60.1 DEG C, repeatedly determines optimum annealing temperature after revision test.Finally determine best reaction conditions: 94 DEG C of 3min; 94 DEG C of 30s; 50 DEG C of 30s; 72 DEG C of 30s, 35 circulations; 72 DEG C of 10min.
Three, multiplex PCR specific test
Multiplexed PCR amplification is carried out according to the PCR reaction system of above-mentioned optimization and the reaction conditions of optimization, different templates is as follows respectively: DHAV-1 cDNA+DHAV-3 cDNA+MDPV cDNA (DHAV-1, DHAV-3 cDNA 1 μ L, MDPV cDNA 2 μ L), extract Muscovy duck parvovirus, duck circovirus, duck plague virus DNA, the cDNA of H5, H9, duck paramyxoviru, duck hepatitis virus.As shown in Figure 1,1-4 has the positive control fragment of 210bp, 375bp and 533bp to result, and all the other all do not have object fragment.Illustrate, primer of the present invention and method have high specific.
Therefore, above-mentioned primer and method can be applicable to qualification unknown sample whether infected duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus:
If obtain the fragment of 210bp, then contain duck hepatitis A virus (HAV) 1 C-type virus C in sample, otherwise then do not have;
If obtain the fragment of 375bp, then contain duck hepatitis A virus (HAV) 3 C-type virus C in sample, otherwise then do not have;
If obtain the fragment of 533bp, then contain Muscovy duck parvovirus in sample, otherwise then do not have;
If obtain the fragment of 210bp and 533bp, then contain duck hepatitis A virus (HAV) 1 C-type virus C and Muscovy duck parvovirus in sample, otherwise then do not have;
If obtain the fragment of 375bp and 533bp, then contain duck hepatitis A virus (HAV) 3 C-type virus C and Muscovy duck parvovirus in sample, otherwise then do not have.
Four, the sensitivity test of multiplex PCR
The nucleic acid concentration recording DHAV-1, DHAV-3 and MDPV with Thermo Scientific NanoDrop 2000 ultramicrospectrophotometer is respectively 52ng/ μ L, 79ng/ μ L and 58ng/ μ L, three to be carried out respectively after 10 times of gradient dilutions by 1:1:2 volume mixture as template, the PCR reaction system optimized according to aforementioned two and the reaction conditions of optimization carry out multiplexed PCR amplification, result as shown in Figure 2,1-5 all has object fragment to expand, and the nucleic acid minimum detectability of the multiple PCR method therefore set up to DHAV-1 is 5.2pg; Being 7.9pg to DHAV-3 minimum detectability, is 5.8pg to the nucleic acid minimum detectability of MDPV.
The assembling of embodiment 3, detection kit
According to the result of study of embodiment 1 and 2, assembling detection kit is with easy to use.
Test kit comprises primer sets, PCR damping fluid and water; Primer sets comprises three pairs of Auele Specific Primers, is primer 1 and 2, primer 3 and 4, primer 5 and 6 respectively, and its concentration is 25 μm of ol/mL (final concentration in PCR reaction system is respectively 0.5 μm of ol/mL, 0.3 μm of ol/mL, 0.7 μm of ol/mL); PCR damping fluid is 2 × Taq PCR Mix.
Other test solutions during PCR detects can situ configuration or direct commodity in use reagent.
Embodiment 4, multiplex PCR detect clinical sample
Get 183 parts of duck pathological material of diseases that laboratory gathers, extract the RNA of duck pathological material of disease, and RNA reverse transcription is obtained cDNA.Respectively using the cDNA that obtains as template, use the test kit of embodiment 3, the PCR reaction system optimized according to embodiment 2 and the reaction conditions of optimization carry out multiplexed PCR amplification.As shown in Figure 3,1-4 is positive control to partial detection, and 7 and 16 have 533bp object fragment, and 11 have 210bp object fragment, and 13 have 375bp object fragment, and all the other are all without other object fragments.7 parts of DHAV-1 are detected in 183 parts of duck pathological material of diseases, infection rate containing DHAV-1 is 3.8% (7/183), 9 parts of DHAV-3 are detected in 183 parts of duck pathological material of diseases, infection rate containing DHAV-3 is 4.9% (9/183), detect 13 parts of MDPV in 183 parts of duck pathological material of diseases, the infection rate containing MDPV is 7.1% (13/183).
Claims (9)
1. duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus multiple PCR detection primer group, it is characterized in that comprising three pairs of Auele Specific Primers, be primer 1 and 2, primer 3 and 4, primer 5 and 6 respectively, they have the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6 respectively.
2. duck hepatitis A virus (HAV) 1 type according to claim 1,3 types and Muscovy duck parvovirus multiple PCR detection primer group, is characterized in that: the mol ratio of described primer 1, primer 2, primer 3, primer 4, primer 5, primer 6 is 0.5-1: 0.5-1: 1: 1: 1: 1.
3. duck hepatitis A virus (HAV) 1 type according to claim 2,3 types and Muscovy duck parvovirus multiple PCR detection primer group, is characterized in that: the mol ratio of described primer 1, primer 2, primer 3, primer 4, primer 5, primer 6 is 1: 1: 1: 1: 1: 1.
4. the non-diagnostic application in pcr amplification of duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus multiple PCR detection primer group described in claim 2, is characterized in that: the annealing temperature of described pcr amplification is 50.0 DEG C-60.1 DEG C.
5. application according to claim 4, is characterized in that: the annealing temperature of described pcr amplification is 50.0 DEG C.
6. duck hepatitis A virus (HAV) 1 type, 3 types and a Muscovy duck parvovirus multiple PCR detection kit, is characterized in that comprising primer sets, PCR damping fluid and water;
Described primer sets comprises three pairs of Auele Specific Primers, primer 1 and 2, primer 3 and 4, primer 5 and 6 respectively, they have the base sequence of sequence table SEQ .ID.No.1 to SEQ.ID.No.6 respectively, its concentration is 25 μm of ol/mL, and the final concentration in PCR reaction system is respectively 0.1 μm of ol/mL-0.5 μm of ol/mL, 0.1 μm of ol/mL-0.5 μm of ol/mL, 0.5 μm of ol/mL-1.0 μm of ol/mL;
Described PCR damping fluid is 2 × Taq PCR Mix.
7. duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus multiple PCR detection kit according to claim 6, is characterized in that: described primer 1 and 2, primer 3 and 4, the final concentration of primer 5 and 6 in PCR reaction system are respectively 0.5 μm of ol/mL, 0.3 μm of ol/mL, 0.7 μm of ol/mL.
8. the non-diagnostic application in pcr amplification of duck hepatitis A virus (HAV) 1 type, 3 types and Muscovy duck parvovirus multiple PCR detection kit described in claim 7, is characterized in that: the annealing temperature of described pcr amplification is 50.0 DEG C-60.1 DEG C.
9. non-diagnostic application according to claim 8, is characterized in that: the annealing temperature of described pcr amplification is 50.0 DEG C.
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| CN109055610A (en) * | 2018-08-15 | 2018-12-21 | 安徽省农业科学院畜牧兽医研究所 | A kind of triple PCR detection method of Muscovy duck parvovirus, 4 type of goose parvovirus and aviadenovirus |
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