CN102925592B - Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection - Google Patents
Fluorescence quantitative RT-PCR (reverse transcription-polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection Download PDFInfo
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- CN102925592B CN102925592B CN 201210493140 CN201210493140A CN102925592B CN 102925592 B CN102925592 B CN 102925592B CN 201210493140 CN201210493140 CN 201210493140 CN 201210493140 A CN201210493140 A CN 201210493140A CN 102925592 B CN102925592 B CN 102925592B
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Abstract
The invention relates to a fluorescence quantitative PCR (polymerase chain reaction) rapid diagnostic kit for specific detection of duck flavivirus infection and an application method thereof. The kit comprises a) an RNA (ribonucleic acid) lysis solution; b) a fluorescence quantitative reaction solution; (c) a duck flavivirus strongly positive quality control product; and (d) a negative quality control product, wherein the fluorescence quantitative reaction solution contains an upstream primer DFV(duck flavivirus)-F, a downstream primer DFV-R and a fluorescent probe DFV-P, and the kit is suitable for rapid quantitative detection of duck flavivirus in clinical applications and scientific research. The kit disclosed by the invention has the following advantages that good specificity, very high accuracy due to double controls of high-specificity amplification of the primers and high-specificity hybridization of the fluorescent probe, and low false positive rate, high sensitivity, fast detection speed, and no post-treatment, no hybridization, electrophoresis, photographing and other processes, and no production of pollution, wherein the detection time is only 2 hours, and the whole process needs about 3 hours in total by considering the extraction of nucleic acid.
Description
Technical field
Fluorescent quantitative PCR rapid diagnosis reagent box and the application method thereof of a kind of specific detection duck flavivirus wild virus infection involved in the present invention, belong to the viral nucleic acid detection field, is applicable to the fast qualitative to duck flavivirus in clinical and scientific research and detects.
Background technology
Since 2010, the ground such as China Shanghai, Zhejiang and Jiangsu have occurred that duck flavivirus infects the death that causes lay eggs degradation and egg duck first.The pest of duck feelings that this cause of disease causes also belong to first at world wide.The same year, the egg drop reduction of unknown cause also appearred in some duckeries in Hubei Province, and clinical manifestation even stops for the degradation of laying eggs, and certain lethality rate is arranged, and cutd open the sick duck spleen of inspection and presented obvious enlargement, and ovary meat becomes, and ovarian follicle is seriously congested, hemorrhage.The outburst of this disease has brought huge financial loss to the raiser.
This seminar has carried out epidemiology survey, pathogen separation, the animal Orthogonal Rotational Regressive Tests of system, order-checking and the analysis of viral portion gene to this disease.Confirm that this disease is to be caused by a kind of new flavivirus, according to the passing rules of viral nomenclature, separate place with reference to virus, by its called after HBJL strain.Preliminary the sequencing results shows that this virus belongs to the ntaya virus group of flaviviridae (Flaviviridae) Flavivirus (Flavivirus) carapuru virus class (Ntaya virus group, NTAV group).
The sub-thread positive chain RNA that the genome of duck flavivirus is non-segmented negative, approximately be comprised of 11000 Nucleotide.When within 2010, our province egg duck cluster breaks out the egg drop reduction epidemic situation, this seminar has carried out massive epidemiology and has investigated and carried out the isolation identification work of cause of disease, with reference to Tembusu virus NS5 gene order autonomous design specific detection primer, carry out SPF egg inoculation and isolate duck flavivirus Hubei strain HBJL detecting positive pathological material of disease.Completed at present the mensuration of duck flavivirus Hubei strain HBJL strain portion gene NS5, and be committed to the American National biomolecule information database (NCBI, accession number: JF423123), with reference to the NS5 gene design of HBJL strain primer:
DFV-F: atgaataaggtggtgaaggtaatgc；
DFV-R： cagattcgtgaaagtgttgagagc；
DFV-P: catgactgttttcccatcacgtcccgg。Product length is 130bp.
Because this disease is the new disease of animal, existing diagnostic method is very limited, mostly be to carry out making a definite diagnosis of this disease by traditional viral separation and Culture (Virus Isolation, VI), polymerase chain TRAP (Polymerase Chain Reaction, PCR).Therefore setting up a kind of quick diagnosis detection method is a problem in the urgent need to address.
Quantitative fluorescent PCR (Fluorogenetic Quatitative PCR, FQ-PCR) method is the technology that the real-time quantitative that grows up the mid-90 in 20th century detects specific nucleic acid, to have increased a fluorescent label DNA probe with high specific on the basis of regular-PCR, by initial point quantitatively and the fluorescence detecting system Real-Time Monitoring accumulate fluorescence intensity and realize nucleic acid is carried out to detection by quantitative.To be used in the world at present virus to detect one of state-of-the-art detection method, with the regular-PCR amplification technique, compare have advantages of incomparable.But this technology has short, high specificity highly sensitive, consuming time, need not gel electrophoresis high-throughput high-level efficiency, the advantage such as detection by quantitative.
Summary of the invention
The fluorescent quantitative PCR rapid diagnosis reagent box and the application method thereof that the object of the present invention is to provide specific detection duck flavivirus that a kind of utilization should fluorescent quantitative PCR technique to infect, this test kit can specially detect duck flavivirus antigen delicately, thereby reaches the purpose of quick diagnosis.
The present invention is that the technical scheme that solves the problems of the technologies described above employing is: the fluorescence quantitative RT-RCR quick diagnosis reagent kit that a kind of specific detection duck flavivirus infects, it is characterized in that this test kit comprises: a) RNA lysate, b) fluorescent quantitation reaction solution, c) duck flavivirus strong positive quality control product, d) negative quality control product, the fluorescent quantitation reaction solution contains upstream primer DFV-F and downstream primer DFV-R and fluorescent probe DFV-P, and upstream primer DFV-F sequence is: atgaataaggtggtgaaggtaatgc 25nt; Downstream primer DFV-R sequence is: cagattcgtgaaagtgttgagagc 24nt; Fluorescent probe DFV-P sequence is: catgactgttttcccatcacgtcccgg 27nt, and that fluorescent probe DFV-P 5 ＇ hold marks is fluorescence report group FAM, that 3 ＇ hold marks is fluorescent quenching group TAMRA.
Press such scheme, the fluorescent quantitation reaction solution is by 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, described upstream primer DFV-F and downstream primer DFV-R are each 0.2 μ M, described fluorescent probe DFV-P is 0.2 μ M.
Press such scheme, the duck flavivirus Jianli strain that described duck flavivirus strong positive quality control product is high titre, it breeds on the duck embryo, after deactivation, obtains, through measuring ELD
50be 10
3.7/ mL.
Press such scheme, described negative quality control product is to gather healthy and, without the duck tissue of cause of disease, 1:5 adds PBS liquid by volume, fully homogenate fragmentation, and centrifugal removal precipitates, and supernatant saves backup.
The application method of the fluorescence quantitative RT-RCR quick diagnosis reagent kit that a kind of specific detection duck flavivirus infects is characterized in that comprising the following steps:
1) extraction of duck flavivirus geneome RNA: add the 1mlRNA lysate respectively in 200 μ l sample to be detected, duck flavivirus strong positive quality control product and negative quality control product, after standing 5min, add 200 μ l chloroforms, draw supernatant after high speed centrifugation, add isopyknic Virahol in supernatant, then carry out ice bath high speed centrifugation precipitation, gained precipitates with after 75% washing with alcohol, with 20 μ l aqua sterilisas, dissolve, gained solution is the RNA template;
2) getting respectively the RNA template that step 1) obtains joins in the fluorescent quantitation reaction solution, carry out the fluorescence intensity discharged in PCR reaction real-time detection reaction process with fluorescent quantitative detector, the PCR reaction obtains duck flavivirus specific amplification goal gene, and described duck flavivirus specific amplification goal gene sequence is: atg aataaggtgg tgaaggtaat gcgaccggga cgtgatggga aaacagtcat ggatgtcatc tcgcgggaag accagagggg aagtggacag gttgtgactt atgctctcaa cactttcacg aatctgt.
Press such scheme, the fluorescent quantitation reaction solution is by 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, described upstream primer DFV-F and downstream primer DFV-R are each 0.2 μ M, described fluorescent probe DFV-P is 0.2 μ M.
Press such scheme, the reaction conditions that carries out the PCR reaction for the fluorescence quantitative RT-RCR detector is:
42 ℃ of reverse transcription 20min
94 ℃ of denaturation 5min
50s, 50 circulations are annealed, extended in 94 ℃ of sex change 10s ← → 60 ℃.
The present invention compared with prior art has the following advantages:
1 specificity is good, and the dual control of high specific hybridization by the amplification of Auele Specific Primer high specific and fluorescent probe, have very high accuracy, and false positive is low;
2 is highly sensitive, and reaction process is monitored in real time by fluorescence detecting system, and fluorescence detecting system has very high detection sensitivity to fluorescent signal;
3 detection speeds are fast, do not need to prepare in addition cDNA, are single stage method, only need 2 hours, add the extraction of nucleic acid, need altogether 3 hours;
4 do not have post-processed, need not hybridization, electrophoresis, the process such as take pictures, and all reagent is all once to increase, and does not need to uncap, and does not produce pollution.
The accompanying drawing explanation
Fig. 1 is that 10 times of gradient dilutions of standard substance carry out the resulting amplification curve of quantitative fluorescent PCR, X-coordinate represents cycle number, and ordinate zou represents fluorescence intensity, and in figure, the straight line of parallel principle X-coordinate represents the fluorescence threshold values, wherein, 1. ~ 7. be followed successively by the standard substance of 10 times of dilutions;
Fig. 2 is that the fluorescent quantitation typical curve of made is as a result carried out in 10 times of dilutions according to standard substance, X-coordinate representative sample extension rate, and ordinate zou represents fluorescence ct value.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be appreciated that these examples only are not used in restriction the scope of protection of present invention for the present invention is described.
The fluorescent quantitative PCR rapid diagnosis reagent box that the specific detection duck flavivirus infects
1. reagent forms:
Trizol RNA lysate is Invitrogen company product; Taq enzyme (5U/ μ L), dNTPs(10mM), MgCI
2(25mM), M-MLV enzyme (50U/ μ L) is all purchased from Promega company; PCR primer pair DFV-F and DFV-R are synthetic by Shanghai Sheng Gong bio-engineering corporation; Fluorescent probe is synthetic by upper sea base health bio-engineering corporation; Preserve in Zhu Youzhe laboratory, duck flavivirus Jianli;
2. reagent preparation:
A) fluorescent quantitation reaction solution: 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, each 0.2 μ M of upstream primer DFV-F and downstream primer DFV-R and fluorescent probe DFV-P 0.2 μ M form; Wherein upstream primer DFV-F sequence is: atgaataaggtggtgaaggtaatgc 25nt; Downstream primer DFV-R sequence is: cagattcgtgaaagtgttgagagc 24nt; Fluorescent probe DFV-P sequence is: catgactgttttcccatcacgtcccgg 27nt, and that fluorescent probe DFV-P 5 ＇ hold marks is fluorescence report group FAM, that 3 ＇ hold marks is fluorescent quenching group TAMRA.
B) the duck flavivirus Jianli strain that duck flavivirus strong positive quality control product is high titre, it breeds on the duck embryo, after deactivation, obtains, through measuring ELD
50be 10
3.7/ mL.
C) negative quality control product: gather healthy and, without each tissue of duck of cause of disease, 1:5 adds PBS liquid by volume, fully homogenate fragmentation, centrifugal removal precipitation, supernatant saves backup.
D) provide reagent for oneself: 75% ethanol of the sterilizing distilled water preparation that chloroform, Virahol, DEPC process.
The using method of the fluorescent quantitative PCR rapid diagnosis reagent box that the specific detection duck flavivirus infects
1, the processing of sample
A) tested sample is tissue sample: get sample 50-100mg to be checked in the homogenizer clean, that sterilizing is also dried, add PBS liquid with the 1:5 volume ratio, fully homogenate, the centrifugal 10min of 4000rpm, get 100 μ L supernatant liquors (each reaction) and proceed in aseptic 1.5mL centrifuge tube, number standby;
B) tested sample is liquid sample (as: whole blood, serum, nose swab etc.), directly gets 100 μ L to proceed in aseptic 1.5mI centrifuge tube, numbers standby.
2, the extraction of tested sample rna
Get the 1.5ml centrifuge tube of n sterilizing, wherein n be the test sample number with duck flavivirus Jianli strain strong positive, contrast and negative quality control product contrast and; Every pipe adds respectively tested sample, duck flavivirus Jianli strain positive control and each 100 μ L of negative quality control product contrast, then adds respectively 500 μ L RNA lysates, fully mixes standing 5min; Add 100 μ L chloroforms, vibration mixes again, 4 ℃ of centrifugal 15min of 12000rpm; Draw supernatant to another centrifuge tube, add isopyknic Virahol, ice bath 15min, 4 ℃ of centrifugal 10min of 12000rpm; Abandon most supernatant, add 1ml 75% ethanol, mix, 4 ℃ of centrifugal 10min of 12000rpm; Thoroughly abandon supernatant.After precipitation Air drying 5min, be dissolved in the sterilizing distilled water that 20 μ L DEPC processed, obtain the RNA template;
3, quantitative fluorescent PCR reaction
Get respectively each 19 μ L of fluorescent quantitation reaction solution, get each 1 μ L of RNA template of the 3rd step gained, add respectively different PCR reaction tubess, detect at the enterprising performing PCR of quantitative fluorescent PCR reaction instrument.Cycling condition is: 94 ℃ of 10s of 94 ℃ of 5min of 42 ℃ of 20min
← →50 circulations of 60 ℃ of 50s amplifications.The program setting of fluoroscopic examination is carried out when the second step of each circulation finishes, and the detection wavelength is 530nm;
The PCR reaction obtains duck flavivirus specific amplification goal gene, and described duck flavivirus specific amplification goal gene sequence is: atg aataaggtgg tgaaggtaat gcgaccggga cgtgatggga aaacagtcat ggatgtcatc tcgcgggaag accagagggg aagtggacag gttgtgactt atgctctcaa cactttcacg aatctgt.
4, result judgement
A) the interpretation of result condition is set
After loop ends, use instrument to carry the software analysis detected result.Circulation thresholding (Threshold Cycle, Ct) setting principle is adjusted according to the noise of instrument situation, and the vertex with Ct value line just above normal negative sample amplification curve is as the criterion;
B) quality control standard
Negative control is without the Ct value and without amplification curve;
Typical amplification curve should and appear in 20 left and right in the Ct value of positive control;
C) result is judged
Negative findings is judged: without the Ct value, and without typical amplification curve;
Positive findings is judged: the Ct value is less than 30, and typical amplification curve occurs;
The experiment gray area: Ct value is greater than 35, and occurs that the sample of typical amplification curve advises reforming.The result of reforming the Ct value occurs and typically the amplification curve person is positive, otherwise negative.
Experimental example 3
The application of the fluorescent quantitative PCR rapid diagnosis reagent box that the specific detection duck flavivirus infects
1, sensitivity test
Carry out (10 of 10 times of gradient dilutions with standard substance
3.7~ 10
-7.7eLD
50/ mL) as template, on quantitative real time PCR Instrument, detect, obtain PCR in real time amplification curve and typical curve and see respectively accompanying drawing 1 and accompanying drawing 2.Detecting minimum virus quantity is 10
-2.7eLD
50/ mL.
2, specific test
The fluorescent quantitative PCR rapid diagnosis reagent box that adopts the specific detection duck flavivirus to infect carries out specific test to bird flu, avian leukosis, avian escherichia coli, Salmonellas, riemerella anatipestifer, newcastle disease, egg-decreasing syndrome etc., all negative to the detected result of above-mentioned 7 kinds of viruses.In addition, all negative to the various tissue detection results of water and duck.Above this test kit of sufficient proof as a result has good specificity in the detection of clinical sample.
3, replica test
The duck flavivirus sample of getting 3 parts of different virus levels organized between repeatability detect, obtain table 1, calculate to such an extent that the between-group variation factor beta value of repeated detection is respectively 4.4%, 4.8%, 3.7%, all be less than 5%, prove that this detection method has good specificity.
Between the group of table 1 sample, repeatability detects the virus titer result
A: circulation thresholding
B: mean CT-number.
Sequence table
<110 > Animal Husbandry and Veterinary Inst., Hubei Academy of Agricultural Sciences (C
<120 > a kind of specific detection duck flavivirus infects fluorescence quantitative RT-RCR quick diagnosis reagent kit and application method thereof
<160>4
<210>1
<211>130
<212>DNA
<213 > duck flavivirus (Duck Flavivirus)
<400>1
atg aataaggtgg tgaaggtaat gcgaccggga cgtgatggga aaacagtcat ggatgtcatc tcgcgggaag accagagggg aagtggacag gttgtgactt atgctctcaa cactttcacg aatctgt 70
<210>2
<211>25
<212>DNA
<213 > artificial sequence
<220>
<223 > according to the design of duck flavivirus nucleotide sequence comparison, for detection of the specific primer sequence of duck flavivirus wild virus infection
<400>2
<210>3
<211>24
<212>DNA
<213 > artificial sequence
<220>
<223 > according to the design of duck flavivirus nucleotide sequence comparison, for detection of the specific primer sequence of duck flavivirus wild virus infection
<400>3
cagattcgtgaaagtgttgagagc 24
<210>4
<211>27
<212>DNA
<213 > artificial sequence
<220>
<223 > according to the design of duck flavivirus nucleotide sequence comparison, for detection of the specificity fluorescent probe of duck flavivirus wild virus infection, that 5' holds mark is fluorescence report group FAM, and that 3' holds mark is fluorescent quenching group TAMRA;
<400>4
catgactgttttcccatcacgtcccgg 27
Claims (4)
1. the fluorescence quantitative RT-RCR quick diagnosis reagent kit that a specific detection duck flavivirus infects, it is characterized in that this test kit comprises: a) RNA lysate, b) fluorescent quantitation reaction solution, c) duck flavivirus strong positive quality control product, d) negative quality control product, the fluorescent quantitation reaction solution contains upstream primer DFV-F and downstream primer DFV-R and fluorescent probe DFV-P, and upstream primer DFV-F sequence is: atgaataaggtggtgaaggtaatgc 25nt; Downstream primer DFV-R sequence is: cagattcgtgaaagtgttgagagc 24nt; Fluorescent probe DFV-P sequence is: catgactgttttcccatcacgtcccgg 27nt, and that fluorescent probe DFV-P 5 ＇ hold marks is fluorescence report group FAM, that 3 ＇ hold marks is fluorescent quenching group TAMRA.
2. the fluorescence quantitative RT-RCR quick diagnosis reagent kit infected by specific detection duck flavivirus claimed in claim 1, is characterized in that the fluorescent quantitation reaction solution is by 1 * PCR Buffer, MgCl
24mM, dNTPs 0.5mM, M-MLV enzyme 50U, Taq enzyme 2U, upstream primer DFV-F and downstream primer DFV-R are each 0.2 μ M, fluorescent probe DFV-P is that 0.2 μ M forms.
3. the fluorescence quantitative RT-RCR quick diagnosis reagent kit infected by specific detection duck flavivirus claimed in claim 1, it is characterized in that the duck flavivirus Jianli strain that described duck flavivirus strong positive quality control product is high titre, it breeds on the duck embryo, after deactivation, obtains, through measuring ELD
50be 10
3.7/ mL.
4. the fluorescence quantitative RT-RCR quick diagnosis reagent kit infected by specific detection duck flavivirus claimed in claim 1, it is characterized in that described negative quality control product be gather healthy and without the duck tissue of cause of disease, 1:5 adds PBS liquid by volume, fully homogenate fragmentation, centrifugal removal precipitation, supernatant saves backup.
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| CN103320541B (en) * | 2013-07-11 | 2016-06-08 | 广西壮族自治区兽医研究所 | Duck flavivirus and duck plague virus duplex RT-PCR detection kit |
| CN106399584B (en) * | 2016-08-23 | 2019-11-19 | 中国水产科学研究院珠江水产研究所 | A kind of mandarin fish infectious spleen and kidney necrosis virus inactivation quick testing reagent kit and detection method |
| CN110846441B (en) * | 2019-12-20 | 2023-04-11 | 浙江省淡水水产研究所 | Specific primer, probe and rapid detection kit for detecting macrobrachium flaviviridae-1 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102220290A (en) * | 2011-05-25 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Cell culture method for duck flavivirus |
| CN102397539A (en) * | 2011-11-25 | 2012-04-04 | 广东省农业科学院兽医研究所 | Duckling flavivirus disease inactivated vaccine and preparation method thereof |
| CN102533668A (en) * | 2010-12-20 | 2012-07-04 | 中国农业科学院上海兽医研究所 | Duck flavivirus, and vaccine and kit thereof |
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| CN102533668A (en) * | 2010-12-20 | 2012-07-04 | 中国农业科学院上海兽医研究所 | Duck flavivirus, and vaccine and kit thereof |
| CN102220290A (en) * | 2011-05-25 | 2011-10-19 | 福建省农业科学院畜牧兽医研究所 | Cell culture method for duck flavivirus |
| CN102397539A (en) * | 2011-11-25 | 2012-04-04 | 广东省农业科学院兽医研究所 | Duckling flavivirus disease inactivated vaccine and preparation method thereof |
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