CN103305633A - Multiplex-fluorescence PCR (Polymerase Chain Reaction) detection kit and application thereof - Google Patents

Multiplex-fluorescence PCR (Polymerase Chain Reaction) detection kit and application thereof Download PDF

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CN103305633A
CN103305633A CN2013102382311A CN201310238231A CN103305633A CN 103305633 A CN103305633 A CN 103305633A CN 2013102382311 A CN2013102382311 A CN 2013102382311A CN 201310238231 A CN201310238231 A CN 201310238231A CN 103305633 A CN103305633 A CN 103305633A
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pcr
fever virus
virus
datum hole
probe
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CN103305633B (en
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吴忠华
郑伟
吕沁风
杨永耀
朱旭平
熊磊
高旭光
杨双彪
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ZHEJIANG INTERNATIONAL TRAVEL HEALTHCARE CENTER
Shanghai ZJ Bio Tech Co Ltd
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ZHEJIANG INTERNATIONAL TRAVEL HEALTHCARE CENTER
Shanghai ZJ Bio Tech Co Ltd
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Abstract

The invention relates to the technical field of biological detection, and in particular relates to a primer, a probe and a kit used for detecting dengue fever, chikungunya and yellow fever virus through a single tube by the multiplex-fluorescence PCR (Polymerase Chain Reaction). The primer and probe used for detecting the dengue fever, chikungunya and yellow fever virus through the multiplex-fluorescence PCR have nucleotide sequences as shown in SEQ ID NO: 1-9; the multiplex PCR detection kit for detecting the dengue fever, chikungunya and yellow fever virus comprises multiplex-fluorescence PCR detection mixed liquid which contains the primer and probe mentioned above. The kit, primer and probe can quickly detect the dengue fever virus, chikungunya virus and yellow fever virus, have the advantages of being simple and convenient to operate, high in sensitivity and outstanding in specificity, and can timely find out and confirm a suspect case so as to improve the monitoring level on such diseases.

Description

A kind of multiple fluorescence PCR detection reagent box and application thereof
Technical field
The present invention relates to technical field of biological, be specifically related to a kind of singapore hemorrhagic fever, datum hole Kenya and yellow fever virus detection and detect primer, probe, test kit and application thereof with multiple fluorescence PCR.
Background technology
Dengue fever virus is small-sized flavivirus, belongs to yellow fever virus and belongs to, and can cause a series of clinical symptom, includes the hemorrhagic shock syndrome of life danger and more rare with decline acute hepatitis with encephalopathic of liver.Be communication media with Aedes aegypti and Aedes albopictus, be widely current in global tropical and semi-tropical more than 60 countries and regions, approximately surpassing 100,000,000 people every year is infected, mortality ratio is up to 50%, people more than 2,500,000,000 is on the hazard, and the propagation of this virus has become the torrid zone, the serious public health problem in subtropical zone.Mortality ratio at the most countries singapore hemorrhagic fever is approximately 5%, occurs in mostly among children and the person between twenty and fifty.
Datum hole Kenya virus belongs to the alphavirus of Togaviridae, and in the disease popularity area, this virus can be present in the multiple animal bodies such as grivet, baboon, chimpanzee, ox, horse, pig, rabbit, and infected animal host and patient are contagium.This virus is propagated by infected mosquito bite, can cause a kind of rare viral infectious---datum hole Kenya heat.The case fatality rate of datum hole Kenya heat is not high, except the minority gerontal patient because of health weak and dead, adult the infected almost do not have death.This disease generally continues five to seven days, and regular meeting causes that serious arthrodynia makes the people lose ability, and this situation finally can be recovered, but has an intense pain and the characteristics slowly recovered can obviously influence people's orthobiosis and work.2006 India and South Asia once in the groove, more than 1,250,000 people infect datum hole Kenya fever virus.In September, 2007, North of Italy has been circulated a notice of to be broken out by the datum hole Kenya heat that an example input case causes.Datum hole Kenya heat is sharply recovered in recent years and has been enlarged territorial scope, has outstandingly shown us to the vulnerability of insect-borne new transmissible disease, and has emphasized the importance as the Sustainable Control planning of hygienic safety fundamental.
Yellow fever virus belongs to flaviviridae, and this virus is one of minimum outer RNA viruses of people, can cause human acute viral transmissible diseases such as yellow jack, also is that first is proved to be by the vector-borne disease of mosquito class.This virus mainly is distributed in South America and area, Africa, and adding up relates to population more than 900,000,000.Estimate that the whole world has 200,000 people to infect this disease every year, 30,000 people's death.1907 after smallpox, the plague, cholera by at that time " International Sanitary Convention " classify the international quarantine transmissible disease as.In 20 years, because the crowd adds the influence of factors such as deforestation, urbanization, movement of population and climate change to the immunity degradation of disease infection, this disease case is increase trend in the past.At present Africa, South America yellow jack are propagated and are become active again, add the convenient of the frequent and vehicles of associating between the world today, and the successor of yellow jack and the danger of going to popular district to infect yellow jack are all strengthening.For this reason, the World Health Organization has called government concerned, department and agency to take action to struggle with yellow jack.Ground such as China Fujian, Guangdong, Hainan exist this sick communication media, in case infect potential popular danger are arranged.So keep very necessary to watching out for of yellow jack in China.
Fluorescent RT-PCR technology is late nineteen nineties in last century new developing technology, and it has fast, specificity, highly sensitive, advantage such as cost is low relatively, is widely used in all respects of clinical detection.Developed at present and utilized fluorescent quantitative PCR technique respectively at above-mentioned three kinds of quick detection kit that virus is carried out, but owing to be single detection, three kinds of viruses detect, not only bothered but also cost of idleness.Therefore on the basis of fluorescent RT-PCR technology, set up the method that the single tube multiple fluorescence PCR detects singapore hemorrhagic fever, datum hole Kenya and yellow fever virus simultaneously and have very important significance.
Summary of the invention
The object of the present invention is to provide a kind of singapore hemorrhagic fever, datum hole Kenya and yellow fever virus multiple fluorescence PCR detection reagent box and preparation method thereof, detection method, can be used for single tube synchronized and detect singapore hemorrhagic fever, datum hole Kenya and yellow fever virus, for clinical suspicious infected patient is carried out etiology differential diagnosis.
The multiple fluorescence PCR that the present invention at first discloses a kind of singapore hemorrhagic fever, datum hole Kenya and yellow fever virus detects with primer and probe, described primer and probe composed as follows:
Dengue fever virus
Dengue fever virus upstream primer: 5 '-TGGGAGAGACCAGAGATCCTG-3 ' (SEQ ID NO:1)
Dengue fever virus downstream primer: 5 '-ATTCAACAGCACCATTCCATTTTC-3 ' (SEQ ID NO:2)
Dengue fever virus probe: 5 '-fluorescence report group-TCTCTACAGCATCATT-fluorescent quenching group-3 ' (SEQ ID NO:3);
Datum hole Kenya virus
Datum hole Kenya virus upstream primer: 5 '-ACGAGGCAAGTTGTCTATTATGAG-3 ' (SEQ ID NO:4)
Datum hole Kenya virus downstream primer: 5 '-GGTAGAGCGTTGACCCTACTG-3 ' (SEQ ID NO:5)
Datum hole Kenya virus probe: 5 '-fluorescence report group-AACAGCACACGGTCGCACGGTT-fluorescent quenching group-3 ' (SEQ ID NO:6);
Yellow fever virus
Yellow fever virus upstream primer: 5 '-TGCTAAGCTGTGAGGCAGTG-3 ' (SEQ ID NO:7)
Yellow fever virus downstream primer: 5 '-CTAGACCCCGTCTTTCTACCAC-3 ' (SEQ ID NO:8)
Yellow fever virus probe: 5 '-fluorescence report group-ACAGCCGACCTCCAGGTTGCGA-fluorescent quenching group-3 ' (SEQ ID NO:9).
Singapore hemorrhagic fever of the present invention, datum hole Kenya and yellow fever virus detect and specifically comprise three pairs of primers and each self-corresponding probe thereof with primer, increase, detect dengue fever virus, datum hole Kenya virus and yellow fever virus respectively.
Probe of the present invention be designed to prior art, probe one end is marked with the fluorescence report group, the probe the other end is marked with the fluorescent quenching group.Preferably, the fluorescence report group of described dengue fever virus probe mark is the FAM fluorophor, and the fluorescence report group of datum hole Kenya virus probe mark is the HEX fluorophor, and the fluorescence report group of yellow fever virus probe mark is Cal Red 610 fluorophors; Described fluorescent quenching group is BHQ1.
Second aspect present invention discloses a kind of singapore hemorrhagic fever, datum hole Kenya and yellow fever virus multiple PCR detection kit, comprises multiple fluorescence PCR and detects mixed solution, and described multiple fluorescence PCR detects in the mixed solution and contains aforesaid primer and probe.
More excellent, described multiple fluorescence PCR detects mixed solution and also contains RT-PCR MIX and H 2O.
Multiple fluorescence PCR of the present invention detects consisting of of mixed solution: dengue fever virus upstream primer 0.5 μ l/test; Dengue fever virus downstream primer 0.5 μ l/test; Dengue fever virus probe 0.1 μ l/test; Datum hole Kenya virus upstream primer 0.5 μ l/test; Datum hole Kenya virus downstream primer 0.5 μ l/test; Datum hole Kenya virus probe 0.1 μ l/test; Yellow fever virus upstream primer 0.5 μ l/test; Yellow fever virus downstream primer 0.5 μ l/test; Yellow fever virus probe 0.1 μ l/test; RT-PCR MIX 12.5 μ l/test; Distilled water 3.2 μ l/test.
More excellent, described singapore hemorrhagic fever, datum hole Kenya and yellow fever virus multiple PCR detection kit also contain RT-PCR enzyme, negative control product (DEPC-H 2O) at least a and in the positive reference substance.
Positive control of the present invention comprises the dengue fever virus plasmid that contains purpose extension increasing sequence shown in the SEQ ID NO:10, the datum hole Kenya virus particle that contains purpose extension increasing sequence shown in the SEQ ID NO:11, and the yellow fever virus plasmid that contains purpose extension increasing sequence shown in the SEQ ID NO:12.
SEQ ID NO:10 sequence is
5’-tgggagagaccagagatcctgctgtctcctcagcatcattccaggcacagaacgccagaaaatggaatggtgctgttgaat-3’
SEQ ID NO:11 sequence is
5’-acgaggcaagttgtctattat?gagagggaaaaagctaaaaccgtgcgaccgtgtgctgttctcagtagggtcaacgctctacc-3’
SEQ ID NO:12 sequence is
5’-tgctaagctgtgaggcagtgcaggctgggacagccgacctccaggttgcgaaaaacctggtttctgggacctcccaccccagagtaaaaagaacggagcctccgctaccaccctcccacgtg?gtggtagaaagacggggtctag-3’
More excellent, the PCR detection architecture of singapore hemorrhagic fever of the present invention, datum hole Kenya and yellow fever virus detection kit fluorescent PCR method is as follows:
Multiple fluorescence PCR detects mixed solution 19 μ l
RT-PCR enzyme 1 μ l
Product to be tested 5 μ l
Reaction totally is 25 μ l.
In the PCR system of test kit of the present invention, described product to be tested is sample nucleic acid extraction liquid, positive control or negative control (DEPC-H 2O).
In the described positive reference substance, singapore hemorrhagic fever plasmid, datum hole Kenya virus particle and yellow fever virus plasmid can be packed separately, also can be hybrid packed.The concentration of singapore hemorrhagic fever plasmid is 5 * 10 3~5 * 10 7Copy/ml; The concentration of datum hole Kenya virus particle is 5 * 10 3~5 * 10 7Copy/ml; The concentration of yellow fever virus plasmid is 5 * 10 3~5 * 10 7Copy/ml.
The preparation method of sample nucleic acid extraction liquid of the present invention can be with reference to existing viral RNA extractive technique, specifically can adopt following method specifically to prepare: to get 100 μ l samples and place the 1.5ml centrifuge tube, add 300 μ l lysates (Trizol), add 100 μ l chloroforms again, vibration mixing 5sec or put upside down mixing 15 times on the vortex mixer; The centrifugal 15min of 13000rpm draws supernatant liquid, adds the equal-volume Virahol, puts upside down the mixing room temperature and leaves standstill 10min, and the centrifugal 10min of 13000rpm removes supernatant gently; Add 700 μ l, 75% ethanol, put upside down washing, the centrifugal 5min of 13000rpm, remove supernatant gently, be inverted on the thieving paper, abandon the centrifugal 5sec of most liquid or 4000rpm, residual liquid on the tube wall is thrown to the pipe bottom, blots liquid with micro sample adding appliance as far as possible, add 20 μ l DEPC-H 2O dissolves RNA, namely obtains sample nucleic acid extraction liquid.
The pcr amplification program of singapore hemorrhagic fever of the present invention, datum hole Kenya and yellow fever virus multiple PCR detection kit is: 45 ℃ of 10min; 95 ℃ of 15min; 95 ℃ of 15sec and 60 ℃ of 60sec alternate cycles 40 times.The single-point fluoroscopic examination is at 60 ℃.
Prepare the sample of sample nucleic acid extraction liquid of the present invention from human body, or virus disseminating media, for example mosquito matchmaker.
The using method of test kit of the present invention may further comprise the steps:
1) preparation of sample nucleic acid extraction liquid;
2) reagent preparation: the configuration multiple fluorescence PCR detects mixed solution, and to wherein adding the RT-PCR enzyme, vibration, centrifugal obtains reaction mixture;
3) reaction mixture application of sample: with step 2) places the PCR pipe, sample nucleic acid extraction liquid, positive reference substance, negative control product are added the different PCR pipes that reaction mixture is housed respectively, obtain sample reaction tubes, negative control reaction tubes, positive reaction pipe;
4) pcr amplification: reaction tubes places on the quantitative fluorescence PCR instrument, and amplification program is set, and carries out pcr amplification;
5) after the PCR reaction finishes, detect the fluorescent signal value of PCR reaction system, fluorescence channel is selected FAM, HEX and Cal Red 610 passages for use.
The test kit quality control: each reference substance of test kit must reach the requirement in the following table 1, otherwise the experiment be considered as invalid.
Table 1 test kit quality standard
Figure BDA00003346150300051
Sample nucleic acid extraction liquid result's judging criterion is as shown in table 2:
Table 2 detected result judging criterion
The multiple fluorescence PCR that the present invention discloses aforementioned singapore hemorrhagic fever, datum hole Kenya and yellow fever virus at last detects the application in preparation singapore hemorrhagic fever, datum hole Kenya disease and/or yellow jack detection reagent with primer and probe, aforementioned singapore hemorrhagic fever, datum hole Kenya and yellow fever virus multiple PCR detection kit.
The present invention relates to three pairs of PCR primers specific amplification dengue fever virus, datum hole Kenya virus and yellow fever virus sequence respectively, three pairs of PCR primers can increase in same PCR reaction tubes simultaneously, realize multiple detection, shorten detection time greatly, and are easy and simple to handle.Auele Specific Primer and and the design of probe guaranteed high conservative and the specificity of primer and probe, avoided no complementary pairing or the situation about increasing of intersecting between two pairs of primers and the probe.The wavelength of fluorescence of three fluorophors that the present invention selects for use differs big and strength of signal is close, has avoided the phase mutual interference between the signal.
Description of drawings
Fig. 1: dengue fever virus sensitivity detection curve figure
Fig. 2: datum hole Kenya moroxydine sensitivity detection curve figure
Fig. 3: yellow fever virus sensitivity detection curve figure
Fig. 4: sample 1 detection curve figure
Fig. 5: sample 2 detection curve figure
Fig. 6: sample 3 detection curve figure
Fig. 7: sample 4 detection curve figure
Fig. 8: sample 5 detection curve figure
Embodiment
Further set forth the present invention below in conjunction with specific embodiment, should be understood that following examples only are used for explanation the present invention and are not used in restriction protection scope of the present invention.
The multiple fluorescence PCR of embodiment 1 singapore hemorrhagic fever, datum hole Kenya and yellow fever virus detects synthesizing with primer and probe
At 5 ' flag F AM fluorophor of dengue fever virus probe, 5 ' mark HEX fluorophor of datum hole Kenya virus probe, 5 ' mark Cal Red610 fluorophor of yellow fever virus probe.The primer of synthetic singapore hemorrhagic fever, datum hole Kenya and yellow fever virus.Probe.
Dengue fever virus:
Dengue fever virus upstream primer: 5 '-TGGGAGAGACCAGAGATCCTG-3 ' (SEQ ID NO:1)
Dengue fever virus downstream primer: 5 '-ATTCAACAGCACCATTCCATTTTC-3 ' (SEQ ID NO:2)
Dengue fever virus probe: 5 ' FAM-TCTCTACAGCATCATT-BHQ1 3 ' (SEQ ID NO:3)
Datum hole Kenya virus:
Datum hole Kenya virus upstream primer: 5 '-ACGAGGCAAGTTGTCTATTATGAG-3 ' (SEQ ID NO:4)
Datum hole Kenya virus downstream primer: 5 '-GGTAGAGCGTTGACCCTACTG-3 ' (SEQ ID NO:5)
Datum hole Kenya virus probe: 5 ' HEX-AACAGCACACGGTCGCACGGTT-BHQ1 3 ' (SEQ ID NO:6)
Yellow fever virus:
Yellow fever virus upstream primer: 5 '-TGCTAAGCTGTGAGGCAGTG-3 ' (SEQ ID NO:7)
Yellow fever virus downstream primer: 5 '-CTAGACCCCGTCTTTCTACCAC-3 ' (SEQ ID NO:8)
Yellow fever virus probe: 5 ' Cal Red 610-ACAGCCGACCTCCAGGTTGCGA-BHQ1 3 ' (SEQ ID NO:9).
Embodiment 2 multiple fluorescence PCRs detect the preparation of mixed solution
With dengue fever virus upstream primer 0.5 μ l/test; Dengue fever virus downstream primer 0.5 μ l/test; Dengue fever virus probe 0.1 μ l/test; Datum hole Kenya virus upstream primer 0.5 μ l/test; Datum hole Kenya virus downstream primer 0.5 μ l/test; Datum hole Kenya virus probe 0.1 μ l/test; Yellow fever virus upstream primer 0.5 μ l/test; Yellow fever virus downstream primer 0.5 μ l/test; Yellow fever virus probe 0.1 μ l/test; RT-PCR MIX 12.5 μ l/test; Process water 3.2 μ l/test mixings are singapore hemorrhagic fever, the datum hole Kenya, and yellow fever virus nucleic acid multiple fluorescence PCR detects mixed solution.
The sensitivity analysis of embodiment 3 singapore hemorrhagic fever, datum hole Kenya and yellow fever virus multiple PCR detection kit
1, experimental technique
1) preparation of sample:
The construction process of dengue fever virus plasmid: adopt the PCR method that the nucleic acid of dengue fever virus sample is increased, obtain to contain the nucleic acid fragment of purpose extension increasing sequence shown in the SEQ ID NO:10, primer sequence is shown in SEQ ID NO:1-2.Behind the fragment purification with amplification, be cloned on the pMD8-T carrier by TA, identify that through order-checking the correct recombinant vectors of sequencing result is transformed into DH5 α, amplification obtains the dengue fever virus plasmid.
The construction process of datum hole Kenya virus particle: adopt the PCR method that the nucleic acid of datum hole Kenya Virus Sample is increased, obtain to contain the nucleic acid fragment of purpose extension increasing sequence shown in the SEQ ID NO:11, primer sequence is shown in SEQ ID NO:4-5.Behind the fragment purification with amplification, be cloned on the pMD8-T carrier by TA, identify that through order-checking the correct recombinant vectors of sequencing result is transformed into DH5 α, amplification obtains datum hole Kenya virus particle.
The construction process of yellow fever virus plasmid: adopt the PCR method that the nucleic acid of yellow fever virus sample is increased, obtain to contain the nucleic acid fragment of purpose extension increasing sequence shown in the SEQ ID NO:12, shown in the primer sequence SEQ ID NO:7-8.Behind the fragment purification with amplification, be cloned on the pMD8-T carrier by TA, identify that through order-checking the correct recombinant vectors of sequencing result is transformed into DH5 α, amplification obtains the yellow fever virus plasmid.
Get 1 * 10 7The dengue fever virus plasmid (DFV) of copies/ml is pressed 1:10,1:100,1:1000,1:10000,1:20000 dilution, obtains DFV-S1(1 * 10 6Copies/ml), DFV-S2(1 * 10 5Copies/ml), DFV-S3(1 * 10 4Copies/ml), DFV-S4(1 * 10 3Copies/ml), DFV-S5(5 * 10 2Copies/ml) totally 5 parts as test sample.
Get 1 * 10 71:10,1:100,1:1000,1:10000,1:20000 dilution are pressed in datum hole Kenya virus particle (CHIKV) of copies/ml, obtain CHIKV-S1(1 * 10 6Copies/ml), CHIKV-S2(1 * 10 5Copies/ml), CHIKV-S3(1 * 10 4Copies/ml), CHIKV-S4(1 * 10 3Copies/ml), CHIKV-S5(5 * 10 2Copies/ml) totally 5 parts as test sample.
Get 1 * 10 7The yellow fever virus plasmid (YFV) of copies/ml is pressed 1:10,1:100,1:1000,1:10000,1:20000 dilution, obtains YFV-S1(1 * 10 6Copies/ml), YFV-S2(1 * 10 5Copies/ml), YFV-S3(1 * 10 4Copies/ml), YFV-S4(1 * 10 3Copies/ml), YFV-S5(5 * 10 2Copies/ml) totally 5 parts as test sample.
2) reagent preparation:
Get n * 19 μ l multiple fluorescence PCRs and detect mixed solution and n * 1 μ l RT-PCR enzyme (n is the reaction tubes number), vibration mixing several seconds, centrifugal several seconds of 3000rpm.
3) application of sample:
Get each 20 μ l of above-mentioned each mixed solution and place the PCR pipe, the plasmid after will diluting then adds respectively in the PCR pipe, builds the pipe lid, carries out pcr amplification reaction immediately.
4) pcr amplification:
Reaction tubes places on the quantitative fluorescence PCR instrument, recommends the loop parameter setting:
45 ℃ * 10min; 95 ℃ * 15min; By 95 ℃ * 15sec → 60 ℃ * 60sec, circulate 40 times again; The single-point fluoroscopic examination is at 60 ℃.Reaction system is 25 μ l.
2. detected result:
Table 3 test kit sensitivity detected result
DFV-S1 DFV-S2 DFV-S3 DFV-S4 DFV-S5
+ + + + -
CHIKV-S1 CHIKV-S2 CHIKV-S3 CHIKV-S4 CHIKV-S5
+ + + + -
YFV-S1 YFV-S2 YFV-S3 YFV-S4 YFV-S5
+ + + + -
As shown in Table 1: for the dengue fever virus plasmid, DFV-S1(1 * 10 6Copies/ml) to DFV-S4(1 * 10 3Copies/ml) equal test positive, and DFV-S5(5 * 10 2Copies/ml) detection is negative, shows that the detection sensitivity of test kit detection dengue fever virus can reach 1 * 10 3Copies/ml.
For datum hole Kenya virus particle, CHIKV-S1(1 * 10 6Copies/ml) to CHIKV-S4(1 * 10 3Copies/ml) equal test positive, and CHIKV-S5(5 * 10 2Copies/ml) detection is negative, shows that the detection sensitivity of test kit detection datum hole Kenya virus can reach 1 * 10 3Copies/ml.
For the yellow fever virus plasmid, YFV-S1(1 * 106copies/ml) is to the equal test positive of YFV-S4(1 * 103copies/ml), and YFV-S5(5 * 102copies/ml) detection is negative, shows that the detection sensitivity of test kit detection yellow fever virus can reach 1 * 10 3Copies/ml.
The use checking of embodiment 4 test kits
1, experimental technique
Be accredited as 1 part in 1 part in dengue fever virus sample, 1 part of datum hole Kenya Virus Sample, 1 part in yellow fever virus sample, 1 part of encephalitis b virus and healthy human body sample through other gold standard method (as order-checking, virus culture etc.), number consecutively is sample 1-5.
2) sample process:
Get 100 μ l samples and place the 1.5ml centrifuge tube, add 300 μ l lysates (Trizol), add 100 μ l chloroforms again, vibration mixing 5sec or put upside down mixing 15 times on the vortex mixer; The centrifugal 15min of 13000rpm draws supernatant liquid, adds the equal-volume Virahol, puts upside down the mixing room temperature and leaves standstill 10min, and the centrifugal 10min of 13000rpm removes supernatant gently; Add 700 μ l, 75% ethanol, put upside down washing, the centrifugal 5min of 13000rpm, remove supernatant gently, be inverted on the thieving paper, abandon the centrifugal 5sec of most liquid or 4000rpm, residual liquid on the tube wall is thrown to the pipe bottom, blots liquid with micro sample adding appliance as far as possible, add 20 μ l DEPC-H 2O dissolves RNA, obtains sample nucleic acid extraction liquid.
2) reagent preparation:
Get n * 19 μ l double fluorescent PCR and detect mixed solution and n * 1 μ l RT-PCR enzyme (n is the reaction tubes number), vibration mixing several seconds, centrifugal several seconds of 3000rpm.
3) application of sample:
Get each 20 μ l of above-mentioned each mixed solution and place the PCR pipe, then with sample nucleic acid extraction liquid, DEPC-H 2O, each 5 μ l of positive reference substance add respectively in the PCR pipe, build the pipe lid, carry out pcr amplification reaction immediately.Reaction system is 25 μ l.
4) pcr amplification:
Reaction tubes places on the quantitative fluorescence PCR instrument, recommends the loop parameter setting:
45 ℃ * 10min; 95 ℃ * 15min; By 95 ℃ * 15sec → 60 ℃ * 60sec, circulate 40 times again; The single-point fluoroscopic examination is at 60 ℃.
5) threshold setting:
The threshold setting principle with threshold line just above DEPC-H 2The vertex of O.
6) quality control:
Each reference substance of test kit must reach following requirement, otherwise the experiment be considered as invalid.
Figure BDA00003346150300101
2 test-results:
Table 4 detected result
Figure BDA00003346150300102
Figure BDA00003346150300111
By table 4 data as can be known, the detected result of sample 1 is the dengue fever virus positive; The detected result of sample 2 is datum hole Kenya virus-positive; The detected result of sample 3 is the yellow fever virus positive, and the test kit detected result of sample 4-5 is negative; The test kit detected result is consistent with the clinical detection result, and visible test kit of the present invention has specificity preferably.
Figure IDA00003346150900021
Figure IDA00003346150900031
Figure IDA00003346150900041
Figure IDA00003346150900051
Figure IDA00003346150900061
Figure IDA00003346150900071

Claims (11)

1. the multiple fluorescence PCR of a singapore hemorrhagic fever, datum hole Kenya and yellow fever virus detects with primer and probe, it is characterized in that described primer and probe composed as follows:
Dengue fever virus upstream primer: 5 '-TGGGAGAGACCAGAGATCCTG-3 '
Dengue fever virus downstream primer: 5 '-ATTCAACAGCACCATTCCATTTTC-3 '
Dengue fever virus probe: 5 '-fluorescence report group-TCTCTACAGCATCATT-fluorescent quenching group-3 '
Datum hole Kenya virus upstream primer: 5 '-ACGAGGCAAGTTGTCTATTATGAG-3 '
Datum hole Kenya virus downstream primer: 5 '-GGTAGAGCGTTGACCCTACTG-3 '
Datum hole Kenya virus probe: 5 '-fluorescence report group-AACAGCACACGGTCGCACGGTT-fluorescent quenching group-3 '
Yellow fever virus upstream primer: 5 '-TGCTAAGCTGTGAGGCAGTG-3 '
Yellow fever virus downstream primer: 5 '-CTAGACCCCGTCTTTCTACCAC-3 '
Yellow fever virus probe: 5 '-fluorescence report group-ACAGCCGACCTCCAGGTTGCGA-fluorescent quenching group-3 '.
2. a singapore hemorrhagic fever, datum hole Kenya and yellow fever virus multiple PCR detection kit comprise multiple fluorescence PCR and detect mixed solution, and described multiple fluorescence PCR detects in the mixed solution and contains the described primer of claim 1 and probe.
3. multiple PCR detection kit as claimed in claim 2 is characterized in that, described multiple fluorescence PCR detects mixed solution and also contains RT-PCR MIX and H 2O.
4. multiple PCR detection kit as claimed in claim 2 is characterized in that, described multiple fluorescence PCR detects consisting of of mixed solution: dengue fever virus upstream primer 0.5 μ l/test; Dengue fever virus downstream primer 0.5 μ l/test; Dengue fever virus probe 0.1 μ l/test; Datum hole Kenya virus upstream primer 0.5 μ l/test; Datum hole Kenya virus downstream primer 0.5 μ l/test; Datum hole Kenya virus probe 0.1 μ l/test; Yellow fever virus upstream primer 0.5 μ l/test; Yellow fever virus downstream primer 0.5 μ l/test; Yellow fever virus probe 0.1 μ l/test; RT-PCR MIX12.5 μ l/test; Distilled water 3.2 μ l/test.
5. multiple PCR detection kit as claimed in claim 2 is characterized in that, described multiple PCR detection kit also contains at least a in RT-PCR enzyme, negative control product and the positive reference substance.
6. multiple PCR detection kit as claimed in claim 5, it is characterized in that, described positive control comprises the dengue fever virus plasmid that contains purpose extension increasing sequence shown in the SEQ ID NO:10, the datum hole Kenya virus particle that contains purpose extension increasing sequence shown in the SEQ ID NO:11, and the yellow fever virus plasmid that contains purpose extension increasing sequence shown in the SEQ ID NO:12; It is described that negative control is arranged is DEPC-H 2O.
7. multiple PCR detection kit as claimed in claim 5 is characterized in that, the PCR detection architecture of singapore hemorrhagic fever, datum hole Kenya and yellow fever virus detection kit fluorescent PCR method is as follows:
Multiple fluorescence PCR detects mixed solution 19 μ l
RT-PCR enzyme 1 μ l
Product to be tested 5 μ l
Reaction totally is 25 μ l.
8. multiple PCR detection kit as claimed in claim 7 is characterized in that, described product to be tested is sample nucleic acid extraction liquid, positive reference substance or negative control product.
9. the using method of the non-diagnostic purpose of the described singapore hemorrhagic fever of the arbitrary claim of claim 2-8, datum hole Kenya and yellow fever virus multiple PCR detection kit, step is as follows:
1) preparation of sample nucleic acid extraction liquid;
2) reagent preparation: the configuration multiple fluorescence PCR detects mixed solution, and to wherein adding the RT-PCR enzyme, vibration, centrifugal obtains reaction mixture;
3) reaction mixture application of sample: with step 2) places the PCR pipe, sample nucleic acid extraction liquid, positive reference substance, negative control product are added the different PCR pipes that reaction mixture is housed respectively, obtain sample reaction tubes, negative control reaction tubes, positive reaction pipe;
4) pcr amplification: reaction tubes places on the quantitative fluorescence PCR instrument, and amplification program is set, and carries out pcr amplification;
5) after the PCR reaction finishes, detect the fluorescent signal value of PCR reaction system, fluorescence channel is selected FAM, HEX and Cal Red 610 passages for use.
10. as the using method of test kit as described in the claim 9, it is characterized in that the described amplification program of step 4) is: 45 ℃ of 10min; 95 ℃ of 15min; 95 ℃ of 15sec and 60 ℃ of 60sec alternate cycles 40 times.
11. the multiple fluorescence PCR of the described singapore hemorrhagic fever of claim 1, datum hole Kenya and yellow fever virus detects with primer and probe, the application in preparation singapore hemorrhagic fever, datum hole Kenya disease and/or yellow jack detection reagent of the described singapore hemorrhagic fever of the arbitrary claim of claim 2-8, datum hole Kenya and yellow fever virus multiple PCR detection kit.
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CN115896343A (en) * 2022-07-15 2023-04-04 云南省寄生虫病防治所 Multiplex fluorescence quantitative PCR detection kit and method for important entomopathogenic virus diseases
CN116064952A (en) * 2022-09-29 2023-05-05 首都医科大学附属北京友谊医院 Double-gene primer probe set for detecting yellow fever virus, kit and application

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CN106755565A (en) * 2015-11-20 2017-05-31 广东省疾病预防控制中心 A kind of kit based on fluorescent PCR method joint-detection dengue fever virus and datum hole Kenya virus
CN106755573A (en) * 2016-12-07 2017-05-31 深圳澳东检验检测科技有限公司 Zika virus, dengue fever virus, the RT PCR detection methods of chikungunya fever virus, primer and probe and kit
CN108330210A (en) * 2017-01-18 2018-07-27 广州市疾病预防控制中心 Zika virus, dengue fever virus and chikungunya virus kit for detecting nucleic acid and application thereof
CN108330210B (en) * 2017-01-18 2021-07-30 广州市疾病预防控制中心 Zika virus, dengue virus and chikungunya virus nucleic acid detection kit and application thereof
CN108707693A (en) * 2018-02-13 2018-10-26 连云港出入境检验检疫局检验检疫综合技术中心(江苏国际旅行卫生保健中心连云港分中心、连云港出入境检验检疫局口岸门诊部) A kind of yellow fever virus, chikungunya virus rapid fluorescence PCR detection kit
CN111826463A (en) * 2019-12-31 2020-10-27 深圳市人民医院 Primer probe combination and kit for detecting five important arthropod/rodent-borne viruses and application of primer probe combination and kit
CN115896343A (en) * 2022-07-15 2023-04-04 云南省寄生虫病防治所 Multiplex fluorescence quantitative PCR detection kit and method for important entomopathogenic virus diseases
CN115896343B (en) * 2022-07-15 2024-10-18 云南省寄生虫病防治所 Multiple fluorescent quantitative PCR detection kit and method for important insect-borne viral diseases
CN116064952A (en) * 2022-09-29 2023-05-05 首都医科大学附属北京友谊医院 Double-gene primer probe set for detecting yellow fever virus, kit and application
CN116064952B (en) * 2022-09-29 2023-08-08 首都医科大学附属北京友谊医院 Double-gene primer probe set for detecting yellow fever virus, kit and application

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