CN105779654A - RT-PCR-HRM or PCR-HRM primer, reagent and method for fast distinguishing four kinds of serotype dengue viruses - Google Patents
RT-PCR-HRM or PCR-HRM primer, reagent and method for fast distinguishing four kinds of serotype dengue viruses Download PDFInfo
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- CN105779654A CN105779654A CN201610248324.6A CN201610248324A CN105779654A CN 105779654 A CN105779654 A CN 105779654A CN 201610248324 A CN201610248324 A CN 201610248324A CN 105779654 A CN105779654 A CN 105779654A
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Abstract
The invention discloses an RT-PCR-HRM or PCR-HRM primer, reagent and method for fast distinguishing four kinds of serotype dengue viruses. According to the genomic sequence alignment result of the four kinds of serotype dengue viruses, three primers are designed, RNA is adopted as a template, an LC green dye is added for carrying out a one-step RT-PCR, or a built plasmid containing a dengue virus 280bp basic group is adopted as a template and the LC green dye is added for carrying out PCR amplification, the amplification product is subjected to HRM analysis, and the four kinds of serotype dengue viruses can be obviously distinguished through an HRM standard curve and a peak type figure. The method is easy to operate, the reagent is low in cost, high in detecting sensitivity and reliable in parting result, and the method is an innovative method for detecting and parting the four kinds of serotype dengue viruses.
Description
Technical field
The method that the present invention relates to gene typing, is specifically related to a kind of four kinds of serotype dengue virus of quick differentiation
RT-PCR-HRM or PCR-HRM primer and method.
Background technology
Dengue virus (dengue virus, DV) is tunicary single strand plus RNA virus, belongs to flaviviridae Flavivirus
Carapuru virus, its primary vehicle is Aedes aegypti and Aedes albopictus, is mainly Aedes albopictus in China.It is to cause the mankind
Dengue fever (dengue fever, DF), dengue hemorrhagic fever (dengue hemorrhagic fever, DHF) and Dengue shock are combined
The main pathogen of simulator sickness (dengue shock syndrome, DSS).1779, in Indonesia Jakarta, Bylon was first
Describe this be referred to as joint heat disease, 1869 by internal medicine institute of London imperial family by this sick named dengue fever.
Dengue fever and dengue hemorrhagic fever are widely current more than 60 countries and regions, the particularly southeast in global tropical and subtropical zone
Asia, Pacific Islands and Caribbean, also occurred the little scope caused by introduced cases to infect in Europe and North America
Popular.In 20th century, dengue fever occurred all over the world to be repeatedly very popular, and case load reaches million, the whole world about 2,500,000,000 population
Being threatened by dengue virus infection, there are about 5,000 ten thousand to one hundred million people every year and infect dengue virus, 500,000 people need to be hospitalized for treatment, about
24000 people are dead.Having the ground such as Guangdong, Guangxi, Hainan, Liaoning in the popular area widely of China, epidemic season Hainan is 4～6
Month, Guangdong and Guangxi are August.As far back as 1873, once there is dengue fever in China Xiamen.1928～nineteen twenty-nine, in Guangzhou, tall building
The ground such as door, Hangzhou, Ningbo, Shanghai, Taiwan and Hong Kong are popular.1940, primary disease was popular to Nantong wide geographic area in Shanghai.1942
～1945, primary disease is popular more serious, is not only popular in coastal area, even spreads to interiorly such as Hankow etc..1978 5
First the moon there is dengue fever in dan gulf town, Foshan, and epidemic situation spreads to surrounding rapidly.Summer in 2002, there is dengue fever in Guangzhou
Popular, there is people more than 1000 to fall ill.From in June, 2014, Guangdong Province there occurs that large-scale Outbreak of Dengue Fever is popular, by the end of
Mid-November, Guangdong the whole province dengue fever morbidity example has broken through 40,000 4 thousand examples, has reported death 6 example.In Kaohsiung City, Taiwan Province, cut
Only in mid-November, 2014, year dengue fever morbidity example also breaks through 10,000 examples, reports death 15 example.2015 by the end of
November 03, Taiwan Province reports cases of dengue fever number 29921 example altogether, and wherein Tainan City reaches 21874 examples, Gaoxiong City 7521 example, screen
County 151, east example, remaining counties and cities distributes the state of an illness with being immigration, and the most accumulative death toll is 129 people, the 27 doubtful deaths of example
Pending trial (platform south 9 examples, Kaohsiung 17 example, Pingtung County 1 example), still there are 28 people in Taiwan in intensive care unit is treated.In China's Mainland, cut-off
To on October 17th, 2015, Chaozhou, Guangdong reported cases of dengue fever 1273 example altogether.
Above data shows, in recent years, dengue fever almost has explosive big at Subtropic of China and torrid areas every year
Scope is popular, forms serious social public health problem in locality.
Dengue virus can be divided into four closely-related serotypes of antigenicity, i.e. Dengue 1,2,3,4 type.Various dengue virus
Between the homology of nucleotide sequence be 63%-68%, between each Strain of same serotype, the homology of nucleotide sequence is more than
95%.4 serotypes all can cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome.DV can present two kinds after infecting body
Different states, i.e. primary infection state and secondary infection state.After body primary infection dengue virus, can behave as recessive sense
Dye or general heating, symptom is inconspicuous, simultaneously to homologous virus generation immunity, and possible even lifelong, but to abnormal shape
The protective effect persistent period that virus infects is shorter.It is susceptible to DHF and DSS in second time infects the patient of special-shaped DV, and
Case fatality rate is higher, and its pathogenesis is to rely on the infection effect that antibody strengthens.Distinguish both states for clinical treatment very
Important.Can use owing to there is presently no successful vaccine, also do not have a specific medicine, therefore dengue virus infection
Early diagnosis and typing, for clinical treatment and the disease surveillance of case, Epidemiological study and diseases control measures
Carry out and have very important meaning.
On dengue virus infection laboratory diagnostic method, the research of serological test and molecular biology has been achieved with one
Fixed progress.The qualification of dengue virus and typing, traditional method mainly has virus purification and qualification, hemagglutination inhibition test, benefit
Body binding tests, neutralization test etc..Sensitive cells is cultivated or animal isolated viral remains " goldstandard " of diagnosis, but this side
Method is very time-consuming, and a lot of laboratory lacks condition essential to virus purification of carrying out.On the other hand, special viral antibody is being sent out
Just can detect after sick 3～5 days, serology detection sensitivity in early days is the highest, and poor specificity, complex operation, time-consuming, especially
It is difficult to accurate typing.Along with succeeding in developing of various dengue virus monoclonal antibody, based on dengue virus type specificity monoclonal
The Classification Identification method of antibody, as IiT (IFA), Enzyme-linked Immunosorbent Assay technology (ELISA) are widely used in
Each dengue virus testing laboratory, the whole world, the accurate typing problem of dengue virus has been resolved, but still suffers from and other jaundice
Poison cross reaction problem, and time-consumingly, cost is high, operation complexity.Nineteen ninety, the application round pcr inspection of the reported first such as Deubel
Survey Dengue Virus.1992, Lanciotti etc. designed a set of nest-type PRC primer, utilizes various dengue virus PCR to expand
The difference of product size carrys out typing.Hereafter, in time more than ten years, Chinese and foreign documents occurs in that much about application round pcr inspection
Survey the report of dengue virus, from multitube two-step method to single tube one-step method, be reacted to single tube respectively from four pairs of type-special primer many
Heavily react, but still suffer from needing multipair primer, electrophoresis detection link easily to pollute and cause the problems such as false positive.Although fluorescent PCR
It is difficult to pollute, but needs multipair primed probe, expensive, thus can not extensively apply.
Said method all fail to overcome operation complicated, time-consuming, costly, the shortcoming such as poor reliability, in clinical practice
Apply limited.Therefore, the dengue virus blood that a kind of operation is relatively simple, testing result is reliable and testing cost is cheap it is badly in need of at present
Clear type classifying method.
Summary of the invention
In order to solve the problem of above-mentioned existence, the present invention establishes a kind of quick differentiation different serotypes dengue virus
RT-PCR-HRM or PCR-HRM primer and method, the method operation is simple, quick, testing result is reliable and testing cost is cheap,
Be conducive to popularization and application in clinical practice.
It is an object of the invention to provide three groups of RT-PCR-HRM or PCR-quickly distinguishing different serotypes dengue virus
HRM primer.
Another object of the present invention is to provide the RT-PCR-HRM of a kind of quick differentiation different serotypes dengue virus or
PCR-HRM method.
It is still another object of the present invention to provide a kind of quick differentiation different serotypes dengue virus RT-PCR-HRM or
PCR-HRM reagent.
The technical solution used in the present invention is:
Base position is quickly distinguishing the application in different serotypes dengue virus, and described base position is:
10648th bit base G, the 10671st bit base T in the DV1 type strain genome of the numbered EU848545.1 of Genbank,
10672nd bit base A, the 10675th bit base G；
10638th bit base A, the 10661st bit base C in the DV2 type strain genome of the numbered AF038403.1 of Genbank,
10662nd bit base T, the 10665th bit base G；
10610th bit base G, the 10633rd bit base C, the in the DV3 type strain genome of the numbered M93130.1 of Genbank
10634 bit base T, the 10637th bit base G；
10570th bit base A, the 10593rd bit base T in the DV4 type strain genome of the numbered AY947539.1 of Genbank,
10594th bit base G, the 10597th bit base A.
Genetic fragment is quickly distinguishing the application in different serotypes dengue virus, and described genetic fragment is:
The genetic fragment of 67bp:
5’-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCTACAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:171)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:172)；
5’-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:173)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTACAACATCAATCCAGGCACAG-3’
(SEQ ID NO:174)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAG-3’
(SEQ ID NO:175)；
The genetic fragment of 45bp:
5 '-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:176)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:177)；
5 '-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:178)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:179)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:180).
A kind of RT-PCR-HRM or PCR-HRM primer of quick differentiation different serotypes dengue virus, described primer amplification
Containing following base position in genes of interest fragment out:
10648th bit base G, the 10671st bit base T in the DV1 type strain genome of the numbered EU848545.1 of Genbank,
10672nd bit base A, the 10675th bit base G；
10638th bit base A, the 10661st bit base C in the DV2 type strain genome of the numbered AF038403.1 of Genbank,
10662nd bit base T, the 10665th bit base G；
10610th bit base G, the 10633rd bit base C, the in the DV3 type strain genome of the numbered M93130.1 of Genbank
10634 bit base T, the 10637th bit base G；
10570th bit base A, the 10593rd bit base T in the DV4 type strain genome of the numbered AY947539.1 of Genbank,
10594th bit base G, the 10597th bit base A.
RT-PCR-HRM or the PCR-HRM primer of a kind of quick differentiation different serotypes dengue virus, described primer includes
Primer P1, P2 and P3；
At least one in SEQ ID NO:1～68 of the base sequence of primer P1；
At least one in SEQ ID NO:69～96 of the base sequence of primer P2；
At least one in SEQ ID NO:97～170 of the base sequence of primer P3.
RT-PCR-HRM or the PCR-HRM primer of a kind of quick differentiation different serotypes dengue virus, described primer includes
Primer P1, P2 and P3；
At least one in SEQ ID NO:1～20 of the base sequence of primer P1；
At least one in SEQ ID NO:69～80 of the base sequence of primer P2；
At least one in SEQ ID NO:97～120 of the base sequence of primer P3.
RT-PCR-HRM or the PCR-HRM primer of a kind of quick differentiation different serotypes dengue virus, described primer includes
Primer P1, P2 and P3；
At least one in SEQ ID NO:1～4 of the base sequence of primer P1；
At least one in SEQ ID NO:69～71 of the base sequence of primer P2；
At least one in SEQ ID NO:99～101 of the base sequence of primer P3.
RT-PCR-HRM or the PCR-HRM reagent of a kind of quick differentiation different serotypes dengue virus, this reagent contains
State described primer P1, P2 and P3.
A kind of RT-PCR-HRM or the PCR-HRM method of quick differentiation different serotypes dengue virus, comprises the following steps:
1) preparation of template: extract viral RNA from sample as template, maybe this RNA reverse transcription is gone out cDNA as template, or
Build the plasmid containing HRM typing fragment as template；
2) with extract RNA as template, with primer described above, P1, P2 are carried out RT-PCR reaction；Or with in step 1)
Described cDNA or plasmid are template, with primer described above, P1, P2 are carried out PCR reaction；
3) with positive reference substance as reference, amplified production is carried out HRM analysis；
If the melting temperature of sample is consistent with DV4 positive reference substance, then it is judged to DV4；
If the melting temperature of sample is consistent with DV2 positive reference substance, then it is judged to DV2；
If the melting temperature of sample is consistent with DV3 positive reference substance, then it is judged to DV3；
If the melting temperature of sample is consistent with the positive reference substance of DV1 or DV4 type strain, then it is judged to DV1 or DV4 type strain；
4) differentiation of DV1 and DV4 type strain:
Repeat step 2) operation, except the P1 of quoting therein and P2 being replaced with primer described above to P1, P3, other behaviour
Make constant；With positive reference substance as reference, amplified production is carried out HRM analysis；
If the melting temperature of sample is consistent with DV1 positive reference substance, then it is judged to DV1；
If the melting temperature of sample is consistent with DV4 type strain positive reference substance, then it is judged to DV4 type strain.
Further, step 2) described in the reaction system of RT-PCR be:
RNA template 2 μ l
10 μm ol primer P1 0.4 μ l
10 μm ol primer P2 or P3 0.4 μ l
TaKaRa Ex Taq HS 0.4μl
PrimeScript RT enzyme Mix II 0.4μl
2X One Step RT-PCR Buffer III 10μl
LC green dyestuff 0.5 μ l
RNase Free ddH2O mends to 20 μ l.
Further, step 2) described in the reaction system of PCR be:
Premix Taq™ 25μl
Plasmid template 2 μ l
10 μm ol forward primer P1 0.5 μ l
10 μm ol downstream primer P2/P3 0.5 μ l
LC green dyestuff 0.5 μ l
ddH2O mends to 50 μ l.
Further, step 2) described in the response procedures of RT-PCR be: 42 DEG C of 30min；94 DEG C of denaturations 2min；94
DEG C degeneration 30s, 53 DEG C of annealing 30s, 72 DEG C extend 30s, circulate 35 times；72 DEG C extend 8min eventually.
Further, described PCR response procedures is: 94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 53～55 DEG C of annealing
30s, 72 DEG C extend 30s；Circulate 35 times；72 DEG C extend 8min eventually.
The invention has the beneficial effects as follows:
1) present invention establishes RT-PCR-HRM or the PCR-HRM primer of a kind of quick differentiation different serotypes dengue virus first
And method, utilize the primer of the present invention and the discriminating the four serotypes of dengue that method energy is easy.
2) present invention is simple to operate: add fluorescence saturable dye before only needing RT-PCR or PCR-HRM reaction；Detection speed
Spend fast and high flux: all operations process only needs 2～3 hours, it is not necessary to the cell of virus is cultivated, needed for greatly shortening typing
Time；Expense is low, it is not necessary to specific probe, and the saturable dye cost of each sample is 1 RMB；Accuracy is high, specificity is good,
Reproducible, accurately, quickly, be analyzed with high throughput can be conducive to popularization and application in clinical practice.
3) RT-PCR-HRM or the PCR-HRM primer versatility of the present invention is good, all has the dengue virus of 4 kinds of serotypes very
Good amplification, is favorably improved the efficiency of PCR, reduces virus and differentiates the time of typing.
4) RT-PCR-HRM or the PCR-HRM primer specificity of the present invention is good, and removing can be with the dengue virus of 4 kinds of serotypes
In conjunction with, be not combined with other common flavivirus viral RNAs, specific amplification dengue viral rna, be conducive to improving the present invention couple
The correctness that serotype is analyzed.
Accompanying drawing explanation
Fig. 1 is four kinds of serotype DV cell culture supernatant 67bp RT-PCR product electrophoretograms；
Fig. 2 is four kinds of serotype DV cell culture supernatant 67bp RT-PCR product standardization melting curve figures；
Fig. 3 is four kinds of serotype DV cell culture supernatant 67bp RT-PCR product peak type melting curve figures；
Fig. 4 is respectively containing the electrophoresis of the 67bp PCR primer of the plasmid of genes of interest in DV1, DV2, DV3, DV4 and DV4 type strain
Figure；
Fig. 5 is respectively containing the 67bp PCR primer standardization of the plasmid of genes of interest in DV1, DV2, DV3, DV4 and DV4 type strain
Melting curve figure；
Fig. 6 is respectively containing the 67bp PCR primer peak type of the plasmid of genes of interest in DV1, DV2, DV3, DV4 and DV4 type strain
Melting curve figure；
Fig. 7 is respectively containing the electrophoretogram of the 45bp PCR primer of the plasmid of genes of interest in DV1, DV4 type strain；
Fig. 8 is respectively containing the 45bp PCR primer standardization melting curve figure of the plasmid of genes of interest in DV1, DV4 type strain；
Fig. 9 is respectively containing the 45bp PCR primer peak type melting curve figure of the plasmid of genes of interest in DV1, DV4 type strain；
Figure 10 is containing the typing sequence of 67bp in the plasmid sequencing result of 280bp genes of interest in DV1；
Figure 11 is containing the typing sequence of 67bp in the plasmid sequencing result of 280bp genes of interest in DV2；
Figure 12 is containing the typing sequence of 67bp in the plasmid sequencing result of 280bp genes of interest in DV3；
Figure 13 is containing the typing sequence of 67bp in the plasmid sequencing result of 280bp genes of interest in DV4；
Figure 14 is vertical luxuriant and rich with fragrance biosynthetic containing the typing sequence of 67bp in 280bp plasmid sequencing result in DV4 type strain.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described further, but is not limited thereto.
Embodiment 1: quickly distinguish base position and the genetic fragment of different serotypes dengue virus
(1) present invention is through lot of experiments, finds following base position to can be used for quickly and distinguishes different serotypes Dengue disease
Poison: the 10648th bit base G, the 10671st bit base T, the in the DV1 type strain genome of the numbered EU848545.1 of Genbank
10672 bit base A, the 10675th bit base G；
10638th bit base A, the 10661st bit base C in the DV2 type strain genome of the numbered AF038403.1 of Genbank,
10662nd bit base T, the 10665th bit base G；
10610th bit base G, the 10633rd bit base C, the in the DV3 type strain genome of the numbered M93130.1 of Genbank
10634 bit base T, the 10637th bit base G；
10570th bit base A, the 10593rd bit base T in the DV4 type strain genome of the numbered AY947539.1 of Genbank,
10594th bit base G, the 10597th bit base A.
(2) present invention is through further experimentation, finds following genetic fragment to can be used for quickly and distinguishes different serum
Type dengue virus:
The genetic fragment of 67bp:
5’-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCTACAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:171)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:172)；
5’-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:173)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTACAACATCAATCCAGGCACAG-3’
(SEQ ID NO:174)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAG-3’
(SEQ ID NO:175)；
The genetic fragment of 45bp:
5 '-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:176)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:177)；
5 '-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:178)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:179)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:180).
Embodiment 2 quickly distinguishes RT-PCR-HRM or the PCR-HRM primer of four kinds of serotype dengue virus
The present invention, after designed a large amount of primers are carried out experiment screening, finds that can be used for quickly distinguishing different serotypes steps on
RT-PCR-HRM or the PCR-HRM primer of leather virus, containing following base position in described primer amplification purpose fragment out:
10648th bit base G, the 10671st bit base T in the DV1 type strain genome of the numbered EU848545.1 of Genbank,
10672nd bit base A, the 10675th bit base G；
10638th bit base A, the 10661st bit base C in the DV2 type strain genome of the numbered AF038403.1 of Genbank,
10662nd bit base T, the 10665th bit base G；
10610th bit base G, the 10633rd bit base C, the in the DV3 type strain genome of the numbered M93130.1 of Genbank
10634 bit base T, the 10637th bit base G；
10570th bit base A, the 10593rd bit base T in the DV4 type strain genome of the numbered AY947539.1 of Genbank,
10594th bit base G, the 10597th bit base A.
Further by experiment screening, find that RT-PCR-HRM method is distinguished four kinds by being used in combination of primer P1, P2, P3
The effect of serotype dengue virus is best.
Wherein, at least one in SEQ ID NO:1～68 of the base sequence of primer P1.
At least one in SEQ ID NO:69～96 of the base sequence of primer P2.
At least one in SEQ ID NO:97～170 of the base sequence of primer P3.
Embodiment 3: the method for the RT-PCR-HRM of a kind of quick four kinds of serotype dengue virus of differentiation
1) extraction of RNA in DV:
The dengue virus cell culture supernatant being clinically separated is provided by Guangzhou City No.8 People's Hospital.With sky root disease poison DNA/
RNA extracts test kit (OSR-M202), extracts isolated clinically on the automatic instrument for extracting nucleic acid of TGuide M16 respectively
Dengue viral rna in four kinds of serotype dengue virus cell culture supernatant, as the template of follow-up One step RT-PCR.
2) One step RT-PCR:
Utilize Takara One Step PrimeScript RT-PCR Kit (Perfect Real Time) test kit, interior
Containing TaKaRa Ex Taq HS, PrimeScript RT enzyme Mix II, 2X One Step RT-PCR Buffer III
Deng.The system of 20 μ l: RNA template adds the amount within 5 μ l, generally 2 μ l according to actual concentrations.TaKaRa Ex Taq HS adds
0.4 μ l, upstream and downstream primer respectively adds 0.4 μ l, PrimeScript RT enzyme Mix II and adds 0.4 μ l, 2X One Step RT-
PCR Buffer III 10 μ l, LC green dyestuff adds 0.5 μ l.With RNase Free ddH2O mends to 20 μ l.
Reaction system concrete in the present embodiment is:
RNA template 2 μ l
10 μm ol primer P1 0.4 μ l
10 μm ol primer P2 0.4 μ l
TaKaRa Ex Taq HS 0.4μl
PrimeScript RT enzyme Mix II 0.4μl
2X One Step RT-PCR Buffer III 10μl
LC green dyestuff 0.5 μ l
RNase Free ddH2O mends to 20 μ l.
The sequence of above-mentioned primer P1 is 5 '-AAACAGCATATTGACGCTGG-3 ' (SEQ ID NO:3)；
The sequence of primer P2 is 5 '-CTGTGCCTGGAATGATG-3 ' (SEQ ID NO:69).
RT-PCR response procedures is: 42 DEG C of 30min；94 DEG C of denaturations 2min；94 DEG C of degeneration 30s, 53 DEG C of annealing 30s, 72
DEG C extend 30s, circulate 35 times；72 DEG C extend 8min eventually, and amplified production is analyzed on Rotor-Gene Q instrument.
3) interpretation of result:
Electrophoresis detection: the electrophoretogram of above-mentioned RT-PCR product is shown in Fig. 1, from figure 1 it appears that obtain clinically in the present embodiment
Four kinds of serotype dengue virus can be amplified out the genetic fragment of 67bp, and carry out checking order (by the raw work biological engineering in Shanghai
Company limited completes), sequencing result shows, DV1(dengue virus 1 type), DV2(dengue type 2 virus), DV3(dengue virus 3 type)
67bp fragment all consistent with corresponding type strain, and DV4(dengue virus 4 type that the present embodiment is selected) 67bp fragment with
DV4 type strain exist a base mutation (the 47th bit base G of 67bp fragment has been mutated into A), i.e. SEQ ID NO:174 with
In SEQ ID NO:175 there is base mutation in the 47th bit base.
HRM analyzes:
Above-mentioned RT-PCR product is all carried out on Rotor-Gene Q instrument HRM(high-resolution melting curve analysis technology) point
Analysis, analysis result is shown in Fig. 2 and Fig. 3.
Fig. 2 is four kinds of serotype DV cell culture supernatant 67bp RT-PCR primer standardization melting curve figures；Therefrom
Can be seen that DV1, DV2, DV3 and DV4 melting curve is separated from each other, show that designed primer P1 and P2 is suitable for this 4 kinds of blood
The HRM of clear type DV analyzes, it is possible to distinguish the dengue virus of four kinds of serotypes.
Fig. 3 is four kinds of serotype DV cell culture supernatant 67bp RT-PCR product peak type melting curve figures；Therefrom may be used
To find out that the melting temperature (Tm) of DV1, DV2, DV3 and DV4 is all different, minimum 76.9 DEG C of the melting temperature of DV4, DV1, DV2,
The melting temperature of DV3 is followed successively by 77.6 DEG C, 77.9 DEG C and 78.16 DEG C.
Embodiment 4: the method for the PCR-HRM of a kind of quick four kinds of serotype dengue virus of differentiation
In order to analyze whether primer P1, P2 can distinguish DV1, DV2, DV3, DV4, DV4 type strain, the present embodiment structure further
Build the 280bp plasmid comprising 67bp and 45bp typing fragment, and additionally add the plasmid of a synthetic gene fragment
(DV4 type strain 280bp fragment, comprises 67bp and 45bp typing fragment to sample, is cloned in puc57 plasmid, by Shanghai Li Feisheng
Thing Technology Co., Ltd. synthesizes).
1) preparation of the Plasmid samples containing DV 280bp genes of interest:
With forward primer 5 '-GCWGCCTGTAGCTCC-3 ' (SEQ ID NO:181), downstream primer 5 '-
CTGTGCCTGGAATGATG-3 ' (SEQ ID NO:182) amplification Dengue virus genes 280bp fragment (comprise 67bp and
45bp HRM typing fragment), purification reclaims PCR primer, is connected with Takara pMD-18T vector.
Carrier coupled reaction system (10 μ l):
Reaction condition: 16 DEG C, more than 4h.
Take-70 DEG C of frozen competent cells after thawed on ice, add and connect product 5 l, mix gently, ice bath
30min.42 DEG C of water-bath heat shock 90sec, then put back to rapidly 2min on ice.Add 400 l LB fluid mediums, 37 DEG C of 200r/
After min-220r/min shaken cultivation 45min, to recover the resistance of plasmid.4 DEG C of 4000r/min are centrifuged 5min, supernatant discarded 400
L, is coated with ammonia benzyl flat board after remaining 100 l bacterium solution mixings, cultivates 30min(plate for 37 DEG C and just put), treat that bacterium solution absorbs completely,
Again plate is inverted and cultivates about 12h～16h, occur to single bacterium colony.With sterilizing toothpick picking list bacterium colony in 5ml containing ampicillin
LB fluid medium in, 37 DEG C of 200r/min-220r/min shaken cultivation 12h～16h, be accredited as the positive through bacterium solution PCR.
2) extracting of plasmid:
DV1, DV2, DV3, DV4, DV4 standard is extracted respectively with the TIANprep Mini Plasmid little extraction reagent kit of Kit plasmid
The plasmid of strain.
3) PCR amplification:
The PCR amplification system of DV1, DV2, DV3, DV4, DV4 type strain plasmid is:
Premix Taq™(TaKaRa Taq™ Version 2.0) 25μl
Plasmid template 2 μ l
10 μm ol forward primer P1 0.5 μ l
10 μm ol downstream primer P2 0.5 μ l
LC green dyestuff 0.5 μ l
ddH2O mends to 50 μ l.
The sequence of above-mentioned primer P1 is 5 '-AAACAGCATATTGACGCTGG-3 ' (SEQ ID NO:3)；
The sequence of primer P2 is 5 '-CTGTGCCTGGAATGATG-3 ' (SEQ ID NO:69).
PCR response procedures:
94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 53 DEG C of annealing 30s are (when downstream primer is P2；Anneal when downstream primer is P3
Temperature is 55 DEG C), 72 DEG C extend 30s；Circulate 35 times；72 DEG C extend 8min eventually.
4) interpretation of result:
Electrophoresis detection: the electrophoretogram of above-mentioned pcr amplification product is shown in Fig. 4, figure 4, it is seen that all samples in the present embodiment
The genetic fragment of 67bp can be amplified out.The 280bp plasmid built is checked order (by the raw limited public affairs of work biological engineering in Shanghai
Take charge of), sequencing result is the most correct.
HRM analyzes: above-mentioned PCR primer all carries out on Rotor-Gene Q instrument HRM(high-resolution fusion curve analysis
Technology) to analyze, analysis result is shown in Fig. 5 and Fig. 6.
Fig. 5 is that the 67bp PCR primer standardization of the DV4 type strain plasmid of four kinds of serotype DV plasmids and synthetic is melted
Solution curve figure；There it can be seen that the standardization melting curve of DV2, DV3, DV4 can distinguish well, but DV1 and DV4
The curve blending of type strain is together, it is impossible to effectively distinguished.
Fig. 6 is that the 67bp PCR primer peak typeization of the DV4 type strain plasmid of four kinds of serotype DV plasmids and synthetic melts
Solution curve figure；Same Fig. 5, the peak type melting curve of DV2, DV3, DV4 can distinguish well, and the melting temperature of DV4 is
79.29 ± 0.028 DEG C, the melting temperature of DV2 is 80.52 ± 0.027 DEG C, and the melting temperature of DV3 is 80.93 ± 0.025 DEG C,
The melting temperature of DV1 or DV4 type strain is 80.26 ± 0.038 DEG C, but together with the curve blending of DV1 with DV4 type strain, no
Can effectively be distinguished.
5) differentiation of DV1 and DV4 type strain:
The plasmid of DV1 and DV4 type strain is carried out PCR amplification respectively, except primer used is P1 and P3, other operation all with
Above-mentioned steps 2)；
The sequence of above-mentioned primer P1 is 5 '-AAACAGCATATTGACGCTGG-3 ' (SEQ ID NO:3)；
The sequence of primer P3 is 5 '-GAGACAGCAGGATCTCTG-3 ' (SEQ ID NO:101)；
Electrophoresis detection: the electrophoretogram of above-mentioned pcr amplification product is shown in Fig. 7, it can be seen from figure 7 that DV1 and DV4 type strain sample
The genetic fragment of 45bp can be amplified out.And it is (biological by the raw work in Shanghai to check order amplification genetic fragment out respectively
Engineering Co., Ltd completes), sequencing result is the most correct.
HRM analyzes: above-mentioned PCR primer all carries out on Rotor-Gene Q instrument HRM(high-resolution fusion curve analysis
Technology) to analyze, analysis result is shown in Fig. 8 and Fig. 9.
Fig. 8 is the 45bp PCR primer standardization melting curve figure of DV1, DV4 type strain plasmid；It can be seen that DV1
Be separated from each other with DV4 type strain melting curve, show designed primer P1 and P3 be suitable for these 2 kinds of serotypes DV(DV1 and
DV4) HRM analyzes.
Fig. 9 is the 45bp PCR primer peak type melting curve figure of DV1, DV4 type strain plasmid；It can be seen that DV1
Different with the melting temperature (Tm) of DV4 type strain, the melting temperature of DV4 type strain is 76.13 ± 0.023 DEG C, the melting temperature of DV1
Degree is 76.77 ± 0.031 DEG C, can be well distinguished out.
Figure 10 is the typing sequence figure of corresponding 67bp base in 280bp base fragment sequencing result in DV1；
Figure 11 be in DV2 in 280bp base fragment sequencing result the typing sequence figure of corresponding 67bp base (sequencer map is anti-
To complementary series)；
Figure 12 is the typing sequence figure of corresponding 67bp base in 280bp base fragment sequencing result in DV3；
Figure 13 is the typing sequence figure of corresponding 67bp base in 280bp base fragment sequencing result in DV4；
Figure 14 is the typing sequence of corresponding 67bp base in the 280bp base fragment founding luxuriant and rich with fragrance biosynthetic DV4 type strain
Figure.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
<110>Guangzhou City No.8 People's Hospital of Experimental Animals Supervising Station, Guangdong Prov.
<120>a kind of RT-PCR-HRM or PCR-HRM primer, reagent and the method for four kinds of serotype dengue virus of quick differentiation
<160> 182
<170> PatentIn version 3.5
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<212> DNA
<213>artificial primer
<400> 135
gacagcagga t11
<210> 136
<211> 18
<212> DNA
<213>artificial primer
<400> 136
acagcaggat ctctggtc 18
<210> 137
<211> 17
<212> DNA
<213>artificial primer
<400> 137
acagcaggat ctctggt 17
<210> 138
<211> 16
<212> DNA
<213>artificial primer
<400> 138
acagcaggat ctctgg 16
<210> 139
<211> 15
<212> DNA
<213>artificial primer
<400> 139
acagcaggat ctctg 15
<210> 140
<211> 14
<212> DNA
<213>artificial primer
<400> 140
acagcaggat ctct 14
<210> 141
<211> 13
<212> DNA
<213>artificial primer
<400> 141
acagcaggat ctc 13
<210> 142
<211> 12
<212> DNA
<213>artificial primer
<400> 142
acagcaggat ct 12
<210> 143
<211> 11
<212> DNA
<213>artificial primer
<400> 143
acagcaggat c11
<210> 144
<211> 17
<212> DNA
<213>artificial primer
<400> 144
cagcaggatc tctggtc 17
<210> 145
<211> 16
<212> DNA
<213>artificial primer
<400> 145
cagcaggatc tctggt 16
<210> 146
<211> 15
<212> DNA
<213>artificial primer
<400> 146
cagcaggatc tctgg 15
<210> 147
<211> 14
<212> DNA
<213>artificial primer
<400> 147
cagcaggatc tctg 14
<210> 148
<211> 13
<212> DNA
<213>artificial primer
<400> 148
cagcaggatc tct 13
<210> 149
<211> 12
<212> DNA
<213>artificial primer
<400> 149
cagcaggatc tc 12
<210> 150
<211> 11
<212> DNA
<213>artificial primer
<400> 150
cagcaggatc t11
<210> 151
<211> 16
<212> DNA
<213>artificial primer
<400> 151
agcaggatct ctggtc 16
<210> 152
<211> 15
<212> DNA
<213>artificial primer
<400> 152
agcaggatct ctggt 15
<210> 153
<211> 14
<212> DNA
<213>artificial primer
<400> 153
agcaggatct ctgg 14
<210> 154
<211> 13
<212> DNA
<213>artificial primer
<400> 154
agcaggatct ctg 13
<210> 155
<211> 12
<212> DNA
<213>artificial primer
<400> 155
agcaggatct ct 12
<210> 156
<211> 11
<212> DNA
<213>artificial primer
<400> 156
agcaggatct c11
<210> 157
<211> 15
<212> DNA
<213>artificial primer
<400> 157
gcaggatctc tggtc 15
<210> 158
<211> 14
<212> DNA
<213>artificial primer
<400> 158
gcaggatctc tggt 14
<210> 159
<211> 13
<212> DNA
<213>artificial primer
<400> 159
gcaggatctc tgg 13
<210> 160
<211> 12
<212> DNA
<213>artificial primer
<400> 160
gcaggatctc tg 12
<210> 161
<211> 11
<212> DNA
<213>artificial primer
<400> 161
gcaggatctc t11
<210> 162
<211> 14
<212> DNA
<213>artificial primer
<400> 162
caggatctct ggtc 14
<210> 163
<211> 13
<212> DNA
<213>artificial primer
<400> 163
caggatctct ggt 13
<210> 164
<211> 12
<212> DNA
<213>artificial primer
<400> 164
caggatctct gg 12
<210> 165
<211> 11
<212> DNA
<213>artificial primer
<400> 165
caggatctct g11
<210> 166
<211> 13
<212> DNA
<213>artificial primer
<400> 166
aggatctctg gtc 13
<210> 167
<211> 12
<212> DNA
<213>artificial primer
<400> 167
aggatctctg gt 12
<210> 168
<211> 11
<212> DNA
<213>artificial primer
<400> 168
aggatctctg g11
<210> 169
<211> 12
<212> DNA
<213>artificial primer
<400> 169
ggatctctgg tc 12
<210> 170
<211> 11
<212> DNA
<213>artificial primer
<400> 170
gatctctggt c11
<210> 171
<211> 67
<212> DNA
<213>dengue virus
<400> 171
aaacagcata ttgacgctgg gagagaccag agatcctgct gtctctacag catcattcca ggcacag
<210> 172
<211> 67
<212> DNA
<213>dengue virus
<400> 172
aaacagcata ttgacgctgg gaaagaccag agatcctgct gtctcctcag catcattcca ggcacag
<210> 173
<211> 67
<212> DNA
<213>dengue virus
<400> 173
aaacagcata ttgacgctgg gagagaccag agatcctgct gtctcctcag catcattcca ggcacag
<210> 174
<211> 67
<212> DNA
<213>dengue virus
<400> 174
aaacagcata ttgacgctgg gaaagaccag agatcctgct gtctctacaa catcaatcca ggcacag 67
<210> 175
<211> 67
<212> DNA
<213>dengue virus
<400> 175
aaacagcata ttgacgctgg gaaagaccag agatcctgct gtctctgcaa catcaatcca ggcacag 67
<210> 176
<211> 45
<212> DNA
<213>dengue virus
<400> 176
aaacagcata ttgacgctgg gagagaccag agatcctgct gtctc 45
<210> 177
<211> 45
<212> DNA
<213>dengue virus
<400> 177
aaacagcata ttgacgctgg gaaagaccag agatcctgct gtctc 45
<210> 178
<211> 45
<212> DNA
<213>dengue virus
<400> 178
aaacagcata ttgacgctgg gagagaccag agatcctgct gtctc 45
<210> 179
<211> 45
<212> DNA
<213>dengue virus
<400> 179
aaacagcata ttgacgctgg gaaagaccag agatcctgct gtctc 45
<210> 180
<211> 45
<212> DNA
<213>dengue virus
<400> 180
aaacagcata ttgacgctgg gaaagaccag agatcctgct gtctc 45
<210> 181
<211> 15
<212> DNA
<213>artificial primer
<400> 181
gcwgcctgta gctcc 15
<210> 182
<211> 17
<212> DNA
<213>artificial primer
<400> 182
ctgtgcctgg aatgatg 17
Claims (12)
1. base position is quickly distinguishing the application in different serotypes dengue virus, and described base position is:
10648th bit base G, the 10671st bit base T in the DV1 type strain genome of the numbered EU848545.1 of Genbank,
10672nd bit base A, the 10675th bit base G；
10638th bit base A, the 10661st bit base C in the DV2 type strain genome of the numbered AF038403.1 of Genbank,
10662nd bit base T, the 10665th bit base G；
10610th bit base G, the 10633rd bit base C, the in the DV3 type strain genome of the numbered M93130.1 of Genbank
10634 bit base T, the 10637th bit base G；
10570th bit base A, the 10593rd bit base T in the DV4 type strain genome of the numbered AY947539.1 of Genbank,
10594th bit base G, the 10597th bit base A.
2. genetic fragment is quickly distinguishing the application in different serotypes dengue virus, and described genetic fragment is:
The genetic fragment of 67bp:
5’-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCTACAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:171)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:172)；
5’-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAG-3’
(SEQ ID NO:173)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTACAACATCAATCCAGGCACAG-3’
(SEQ ID NO:174)；
5’-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCTGCAACATCAATCCAGGCACAG-3’
(SEQ ID NO:175)；
The genetic fragment of 45bp:
5 '-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:176)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:177)；
5 '-AAACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:178)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:179)；
5 '-AAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTC-3 ' (SEQ ID NO:180).
3. a RT-PCR-HRM or PCR-HRM primer for quick differentiation different serotypes dengue virus, described primer amplification goes out
Containing following base position in the genes of interest fragment come:
10648th bit base G, the 10671st bit base T in the DV1 type strain genome of the numbered EU848545.1 of Genbank,
10672nd bit base A, the 10675th bit base G；
10638th bit base A, the 10661st bit base C in the DV2 type strain genome of the numbered AF038403.1 of Genbank,
10662nd bit base T, the 10665th bit base G；
10610th bit base G, the 10633rd bit base C, the in the DV3 type strain genome of the numbered M93130.1 of Genbank
10634 bit base T, the 10637th bit base G；
10570th bit base A, the 10593rd bit base T in the DV4 type strain genome of the numbered AY947539.1 of Genbank,
10594th bit base G, the 10597th bit base A.
RT-PCR-HRM or PCR-HRM of a kind of quick differentiation different serotypes dengue virus the most according to claim 3
Primer, described primer includes primer P1, P2 and P3；
At least one in SEQ ID NO:1～68 of the base sequence of primer P1；
At least one in SEQ ID NO:69～96 of the base sequence of primer P2；
At least one in SEQ ID NO:97～170 of the base sequence of primer P3.
RT-PCR-HRM or PCR-HRM of a kind of quick differentiation different serotypes dengue virus the most according to claim 4
Primer, described primer includes primer P1, P2 and P3；
At least one in SEQ ID NO:1～20 of the base sequence of primer P1；
At least one in SEQ ID NO:69～80 of the base sequence of primer P2；
At least one in SEQ ID NO:97～120 of the base sequence of primer P3.
RT-PCR-HRM or PCR-HRM of a kind of quick differentiation different serotypes dengue virus the most according to claim 5
Primer, described primer includes primer P1, P2 and P3；
At least one in SEQ ID NO:1～4 of the base sequence of primer P1；
At least one in SEQ ID NO:69～71 of the base sequence of primer P2；
At least one in SEQ ID NO:99～101 of the base sequence of primer P3.
7. RT-PCR-HRM or the PCR-HRM reagent of a quick differentiation different serotypes dengue virus, it is characterised in that: this examination
Agent contains claim 3～6 arbitrary described primer P1, P2 and P3.
8. RT-PCR-HRM or the PCR-HRM method of a quick differentiation different serotypes dengue virus, it is characterised in that: include
Following steps:
1) preparation of template: extract viral RNA from sample as template, maybe this RNA reverse transcription is gone out cDNA as template, or
Build the plasmid containing HRM typing fragment as template；
2) with the RNA that extracts as template, with claim 3～6 arbitrary described primer, P1, P2 are carried out RT-PCR reaction；Or
Person, with the cDNA described in step 1) or plasmid as template, carries out PCR with the arbitrary described primer of claim 3～6 to P1, P2
Reaction；
3) with positive reference substance as reference, amplified production is carried out HRM analysis；
If the melting temperature of sample is consistent with DV4 positive reference substance, then it is judged to DV4；
If the melting temperature of sample is consistent with DV2 positive reference substance, then it is judged to DV2；
If the melting temperature of sample is consistent with DV3 positive reference substance, then it is judged to DV3；
If the melting temperature of sample is consistent with the positive reference substance of DV1 or DV4 type strain, then it is judged to DV1 or DV4 type strain；
4) differentiation of DV1 and DV4 type strain:
Repeat step 2) operation, except the P1 of quoting therein and P2 being replaced with the arbitrary described primer pair of claim 3～6
P1, P3, other operations are constant；With positive reference substance as reference, amplified production is carried out HRM analysis；
If the melting temperature of sample is consistent with DV1 positive reference substance, then it is judged to DV1；
If the melting temperature of sample is consistent with DV4 type strain positive reference substance, then it is judged to DV4 type strain.
Method the most according to claim 8, it is characterised in that: step 2) described in the reaction system of RT-PCR be:
RNA template 2 μ l
10 μm ol primer P1 0.4 μ l
10 μm ol primer P2 or P3 0.4 μ l
TaKaRa Ex Taq HS 0.4μl
PrimeScript RT enzyme Mix II 0.4μl
2X One Step RT-PCR Buffer III 10μl
LC green dyestuff 0.5 μ l
RNase Free ddH2O mends to 20 μ l.
Method the most according to claim 8, it is characterised in that: step 2) described in the reaction system of PCR be:
Premix Taq™ 25μl
Plasmid template 2 μ l
10 μm ol forward primer P1 0.5 μ l
10 μm ol downstream primer P2/P3 0.5 μ l
LC green dyestuff 0.5 μ l
ddH2O mends to 50 μ l.
11. methods according to claim 8 or claim 9, it is characterised in that: step 2) described in the response procedures of RT-PCR be:
42℃ 30min；94 DEG C of denaturations 2min；94 DEG C of degeneration 30s, 53 DEG C of annealing 30s, 72 DEG C of extension 30s, circulate 35 times；72 DEG C of ends
Extend 8min.
12. according to the method described in claim 9 or 10, it is characterised in that: described PCR response procedures is: 94 DEG C of denaturations
5min；94 DEG C of degeneration 30s, 53～55 DEG C of annealing 30s, 72 DEG C extend 30s；Circulate 35 times；72 DEG C extend 8min eventually.
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| CN104328222A (en) * | 2014-11-17 | 2015-02-04 | 扬州大学 | Kit capable of detecting and parting dengue viruses by reverse transcription PCR and detection method of kit |
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| CN104630388A (en) * | 2014-09-10 | 2015-05-20 | 广州市第八人民医院 | Dengue virus rapid classification identification detection kit |
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