CN105525039A - Amplification method for whole genome aiming at different enterovirus serotypes - Google Patents
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Abstract
The invention discloses an amplification method for a whole genome aiming at different enterovirus serotypes. The method comprises the following steps: extracting RNA of the viruses, carrying out RT-PCR, sequencing and comparison on the VP1 area of the RNA of the viruses by adopting the universal primers 222-224 of the enteroviruses, and determining the serotypes of virus strains by utilizing a molecular biology experimental technology; and designing corresponding primers, namely, designing the primers by utilizing the conservative property of the enteroviruses 5'UTR and 3'UTR, and carrying out corresponding RT-PCR amplification, sequencing and comparison according to the above paired primers till the nucleotide sequence of the whole genome is obtained. The method provided by the invention has the advantages of high amplification speed, as well as simplicity, convenience and feasibility, the cost can be greatly reduced, and the operation is efficient and rapid.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of amplification method of different enterovirus serotype full-length genome.
Background technology
Human enterovirus virus (humanenterovirus, HEV) Picornaviridae enterovirus genus is belonged to, according to current classification, enterovirus is mainly divided into four groups: Human enterovirus virus A, B, C and D group, have serotype more than 100, wherein A group comprises CA group (Coxsackievirus, CV-A) CV-A2 ~ 8, CV-A10, CV-A12, CV-A14, CV-A16 in, EV-A71, CV-A76, CV-A89-CV-A92 and CV-A119; B group comprises all echo, enteric cytopathogenic human orphan virus (echoviruses, E-) and Coxsackie B virus group (CV-B), also has CV-A9 and EV-B69, EV-B73-B75, EV-B77-88, EV-B93, EV-B97-98, EV-B100-101, EV-B106-107 and EV-B110; C group comprises CV-A1, CV-A11, CV-A13, CV-A17, CV-A19-CV-A22, CV-A24, EV-C95, EV-C96, EV-C99, EV-C102, EV-C104, EV-C105, EV-C109, EV-C116-EV-C118, Polio1-3; D group comprises EV-D68, EV-D70, EV-D94, EV-D111 and EV-D120.
Prior art obtains a viral whole genome sequence, first to be increased its VP1 gene by neutralization test or RT-PCR, specify the serotype of enterovirus, then design primer according to the nucleotide sequence of its serotype prototype-strain, a point 6-7 fragment increases respectively and checks order.This method for other viral full-length genome that increases (as mumps virus, Measles virus etc., these virus serotypes only have one) can better result be obtained, enterovirus is because serotype is numerous, nucleotide difference between different serotypes is larger, and difference is also larger between phase homologous serotype, in addition, enterovirus is ubiquity recombination event also, it is more difficult that traditional method is used for the amplification of enterovirus whole genome sequence, proliferation time often reaches 3-6 month, waste time and energy, workload is very large, costly.
Enterovirus causes various diseases as the pathogenic agent of hand foot mouth disease, viral encephalitis, myocarditis etc., it is again RNA viruses, genovariation is very fast, and existing genome amplification method can not be satisfied the demand, and is necessary very much the amplification method improving enterovirus whole genome sequence.
Summary of the invention
Technical problem to be solved by this invention is, overcomes the above defect, provides the amplification method of the different enterovirus serotype full-length genome that a kind of amplification rate is fast, method is simple.
In order to solve the above technical problem, the amplification method of different enterovirus serotype full-length genome of the present invention comprises the following steps:
(1) viral RNA is extracted, the VP1 district of enterovirus universal primer 222-224 to viral RNA is utilized to carry out RT-PCR amplification, then electrophoresis detection is carried out with sepharose, the voltage 120V of 1%, detected result with predict the outcome close, amplification gained Nucleotide object band is checked order, and carry out NCBIBLAST comparison, if enterovirus Reference Strains nucleotide sequence homology is greater than 75% in this inquiry sequence and GenBank database, namely think same serotype, namely obtain the nucleotide sequence of described virus VP 1 district gene simultaneously;
(2) conservative property of enterovirus 5 ' UTR and 3 ' UTR is utilized, a head end upstream primer EV1F(5 ' TTAAAACAGCCTGTGGGTTG3 ' is designed for enterovirus full-length genome) and a tail end downstream primer EV8R(5 ' CACCGAATGCGGAGAATTTA3 '), VP1 district actual nucleotide sequence viral according to step (1) amplification gained again, design the pairing upstream primer EV3F of a pairing downstream primer EV2R and described tail end downstream primer EV8R of a described head end upstream primer EV1F, then RT-PCR amplification is carried out with EV1F-EV2R and EV3F-EV8R respectively, its product with 1% sepharose, 120V carries out electrophoresis detection, if ultraviolet gel imaging instrument detected result has the electrophoretic band close to 2400bp with 5000bp namely to conform to expected results, amplification terminates, and check order with corresponding amplimer,
(3) check order, splice and arrange and check order, until obtain whole genome nucleotide sequence according to step (2) gained nucleotide sequence bamboo product sequencing primer.
Human enterovirus virus (HEV) is the RNA viruses of single-stranded positive, genome is about 7400bp, and only containing an open reading frame (ORF), this ORF is divided into P1, P2 and P3 tri-districts successively, wherein the Structural protein VP1 of P1 district encode viral is to VP4, composition viral capsid; P2 and P3 district is encodes nonstructural proteins 2A, 2B, 2C and 3A, 3B, 3C, 3D respectively.HEV5 ' non-coding region (UTR) is relative conservative with 3 ' non-coding region (UTR).Genotyping result is carried out consistent with neutralization test qualification result according to VP1 region sequence, high compared with other structural protein coding region with serotype dependency, and VP1 albumen is in main virus and factor of determination, it directly determines the antigenicity of virus, therefore, VP1 not only can be used as the foundation of different serotypes classification in enterovirus genus, and can be used as the classification reference do not belonged to together in Picornaviridae.
The present invention is according to the genetic traits of enterovirus own and " walking with primer " method, first carry out RT-PCR(reverse transcription polymerase chain reaction with the VP1 district of universal primer 222-224 to viral RNA) amplification, obtain the actual nucleotide sequence of described virus VP 1 district gene, again based on this, utilize the conservative property of enterovirus 5 ' UTR and 3 ' UTR, design a head end upstream primer EV1F and tail end downstream primer EV8R, again according to the VP1 district actual nucleotide sequence of amplification gained virus, design the pairing upstream primer EV3F of a pairing downstream primer EV2R and tail end downstream primer EV8R of a head end upstream primer EV1F, then RT-PCR amplification is carried out with EV1F-EV2R and EV3F-EV8R respectively, and with " walking with primer " method design sequencing primer, complete the mensuration of increased gene fragment order.The method of the invention obtains a complete enterovirus full-length genome expense and only needs 1/3rd of prior art, time also shortened to 2 weeks from original 3-6 month, and only need three RT-PCR to complete, time saving and energy saving, significantly reduce workload, efficiently can carry out the amplification of different enterovirus serotype strain, for large-scale enterovirus Molecular Epidemic Hygienic monitoring on hands of childhood provides guarantee.
Beneficial effect of the present invention: owing to employing the above technical scheme, the method of the invention has the advantages such as amplification rate is fast, amplification method is simple and easy to do, significantly reduce costs, be a kind of efficiently, the amplification method of different enterovirus serotype full-length genome fast.
Accompanying drawing explanation
Fig. 1 is enterovirus full genome of the present invention amplification schematic diagram;
Fig. 2 is the part VP1 gene electrophorogram of CV-B1, CV-B2, E-33 virus in the present invention's three embodiments;
Fig. 3 is CV-B1, CV-B2, E-33 virus other parts gene fragment electrophorogram in the present invention's three embodiments.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail.
The amplification method of a kind of different enterovirus serotype full-length genome of the present invention, said method comprising the steps of:
(1) viral RNA is extracted, the VP1 district of enterovirus universal primer 222-224 to viral RNA is utilized to carry out RT-PCR amplification, then electrophoresis detection is carried out with sepharose, the voltage 120V of 1%, detected result with predict the outcome close, amplification gained Nucleotide object band is checked order, and carry out NCBIBLAST comparison, if enterovirus Reference Strains nucleotide sequence homology is greater than 75% in this inquiry sequence and GenBank database, namely think same serotype, namely obtain the nucleotide sequence of described virus VP 1 district gene simultaneously;
(2) conservative property of enterovirus 5 ' UTR and 3 ' UTR is utilized, a head end upstream primer EV1F(5 ' TTAAAACAGCCTGTGGGTTG3 ' is designed for enterovirus full-length genome) and a tail end downstream primer EV8R(5 ' CACCGAATGCGGAGAATTTA3 '), VP1 district actual nucleotide sequence viral according to step (1) amplification gained again, design the pairing upstream primer EV3F of a pairing downstream primer EV2R and described tail end downstream primer EV8R of a described head end upstream primer EV1F, then RT-PCR amplification is carried out with EV1F-EV2R and EV3F-EV8R respectively, its product with 1% sepharose, 120V carries out electrophoresis detection, if ultraviolet gel imaging instrument detected result has the electrophoretic band close to 2400bp with 5000bp namely to conform to expected results, amplification terminates, and check order with corresponding amplimer,
(3) check order, splice and arrange and check order, until obtain whole genome nucleotide sequence according to step (2) gained nucleotide sequence bamboo product sequencing primer.
(3) check order, splice and arrange and check order, until obtain whole genome nucleotide sequence according to step (2) gained nucleotide sequence bamboo product sequencing primer.
Virus is isolated from the ight soil of clinical patient, namely after three generations's blind passage cells showed cytopathic alternatively use enterovirus, RT-PCR amplification, order-checking, comparison is carried out with enterovirus universal primer 222-224, identify the serotype of virus, gene order-checking is carried out to the virus of different serotypes.Method is: " walking with primer ", is exemplified below:
Embodiment one: for Coxsackie B virus 1(CV-B1)
(1) RT-PCR is carried out with the VP1 district of enterovirus universal primer 222 (5 ' CICCIGGIGGIAYRWACAT3 ')-224 (GCIATGYTIGGIACICAYRT) to viral RNA, then the sepharose of 1% is used, voltage 120V carries out electrophoresis detection, detected result compares with predicting the outcome, the close (see figure 2) of result, the analysis tool of carrying out similarity system design in Protein Data Bank or DNA database is enclosed within one of NCBIBLAST(American National Biotechnology Information center after order-checking, blast program can rapidly and public data storehouse carry out similarity gene comparision) comparison, this nucleotide sequence and coxsackie virus B1 homology be 92%, according to somatotype standard, can judge that this virus is as Coxsackie B virus 1(CV-B1), current amplification obtains the nucleotide sequence of this virus part VP1 gene in genome,
(2) to hold according to enterovirus genome 5' and the conservative property of 3' end, and the nucleotide sequence of part VP1 gene that step (1) obtains, design primer; Divide two sections, design primer is: EV1F-B12R(is about 2400bp), B13F-EV8R(is about 5000bp) (see table 2), take viral RNA as template, EV1F-B12R(is about 2400bp), B13F-EV8R(is about 5000bp) carry out RT-PCR amplification respectively for primer, be designated as B11 and B12 respectively, system is 30 μ l
2×stepbuffer15μl
Template5μl
dH2O7μl
Enzyme1μl
The each 1 μ l of Primer
RT-PCR optimum configurations is: 50 DEG C of 30min; 94 DEG C of 2min; (94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 2.5min) × 35cycles(circulates), wherein B12 is 72 DEG C of needs 5min times; 72 DEG C of 10min; After terminating RT-PCR, with the sepharose of 1%, 120V carries out electrophoresis detection, and ultraviolet gel imaging instrument detected result has the electrophoretic band close to 2400bp and 5000bp, with the close (see figure 3) that predicts the outcome, can check order;
(3) check order:
The RT-PCR stoste order-checking of B11, respectively first with the order-checking of EV1F and B12R primer, and then designs primer B11r and B12F respectively according to the sequence of sequencing result and checks order;
The RT-PCR stoste order-checking of B12, respectively first with the order-checking of B13F and B18R primer, and then design primer B14F respectively according to the sequence of sequencing result, B14R, B15F, B16F, B16R and B17F check order;
Namely above sequencing result all by with after NCBIBLAST comparison, and is defined as object nucleotide sequence with the corresponding nucleotide sequence homology of CV-B1 or other enterovirus serotype is higher.
(4) nucleotide sequence that above comparison is determined is carried out splicing and be organized into complete whole genome sequence.
The part VP1 gene order (being scribed ss primer) of table 1:CV-B1
Table 2: the order-checking of using in embodiment one and amplimer
Remarks :-: position is uncertain
Embodiment two: for Coxsackie B virus 2(CV-B2)
(1) RT-PCR is carried out with the VP1 district of enterovirus universal primer 222-224 to viral RNA, then electrophoresis detection is carried out with sepharose, the voltage 120V of 1%, detected result compares with predicting the outcome, the close (see figure 2) of result, check order comparison in rear NCBIBLAST, the homology of this nucleotide sequence and CV-B2 is 92%, according to somatotype standard, can judge that this virus is as CV-B2, and obtain the nucleotide sequence of this virus part VP1 gene in genome.
(2) according to the conservative type that enterovirus genome 5' holds and 3' holds, and the nucleotide sequence of the part VP1 gene of step (1) acquisition, design primer; Divide two sections, design primer is: EV1F – B22R(is about 2400bp), B23F-EV8R(is about 5000bp) (see table 4), take viral RNA as template, EV1F-B22R(is about 2400bp), B23F-EV8R(is about 5000bp) for primer carries out RT-PCR respectively, be designated as B21 and B22 respectively, system is 30 μ l
2×stepbuffer15μl
Template5μl
dH2O7μl
Enzyme1μl
The each 1 μ l of Primer
RT-PCR optimum configurations is: 50 DEG C of 30min; 94 DEG C of 2min; Circulate in (94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 2.5min) × 35, wherein B22 is 72 DEG C of needs 5min times; 72 DEG C of 10min; After terminating RT-PCR, with the sepharose of 1%, 120V carries out electrophoresis detection; Ultraviolet gel imaging instrument detected result has the electrophoretic band close to 2400bp and 5000bp, with predict the outcome close (Fig. 3), can check order;
(3) check order:
The RT-PCR stoste order-checking of B21, respectively first with the order-checking of EV1F and B22R primer, and then designs primer B21r and B22F respectively according to the sequence of sequencing result and checks order;
The RT-PCR stoste order-checking of B22, respectively first with the order-checking of B23F and EV8R primer, and then design primer B24R respectively according to the sequence of sequencing result, B25F, B25R and B27F1 check order;
Namely above sequencing result all by with after NCBIBLAST comparison, and is defined as object nucleotide sequence with the corresponding nucleotide sequence homology of CV-B1 or other enterovirus serotype is higher.
(4) nucleotide sequence that above comparison is determined is carried out splicing and be organized into complete whole genome sequence.
The part VP1 gene order (being scribed ss primer) of table 3:CV-B2
Order-checking used by table 4CV-B2 whole genome amplification and amplimer
Remarks :-: position is uncertain
Embodiment three: for Echo virus 33 type (E-33)
(1) RT-PCR is carried out with the VP1 district of enterovirus universal primer 222-224 to viral RNA, then electrophoresis detection is carried out with sepharose, the voltage 120V of 1%, detected result compares with predicting the outcome, the close (see figure 2) of result, check order comparison in rear NCBIBLAST, the homology of this nucleotide sequence and E-33 is 83%, according to somatotype standard, can judge that this virus is as E-33, and obtain the nucleotide sequence of this virus part VP1 gene in genome;
(2) to hold according to enterovirus genome 5' and the conservative type of 3' end, and the nucleotide sequence of part VP1 gene that step (1) obtains, design primer; Divide two sections, design primer is: EV1F – E332R(is about 2400bp), E333F-EV8R(is about 5000bp) (see table 6), take viral RNA as template, EV1F-E332R(is about 2400bp), E333F-EV8R(is about 5000bp) for primer carries out RT-PCR respectively, be designated as E331 and E332 respectively, system is 30 μ l
2×stepbuffer15μl
Template5μl
dH2O7μl
Enzyme1μl
The each 1 μ l of Primer
RT-PCR optimum configurations is: 50 DEG C of 30min; 94 DEG C of 2min; Circulate in (94 DEG C of 30s, 52 DEG C of 30s, 72 DEG C of 2.5min) × 35, wherein E332 is 72 DEG C of needs 5min times; 72 DEG C of 10min; After terminating RT-PCR, with the sepharose of 1%, 120V carries out electrophoresis detection; Ultraviolet gel imaging instrument detected result has the electrophoretic band close to 2400bp and 5000bp, with predict the outcome close (Fig. 3), can check order;
(3) check order:
The RT-PCR stoste order-checking of E331, respectively first with the order-checking of EV1F and E332R primer, and then designs primer E331R and E332F respectively according to the sequence of sequencing result and checks order;
The RT-PCR stoste order-checking of E3322, respectively first with the order-checking of E333F and EV8R primer, and then design primer E334F respectively according to the sequence of sequencing result, E335F1, E335F2, E335R and E336F check order;
Namely above sequencing result all by with after NCBIBLAST comparison, and is defined as object nucleotide sequence with the corresponding nucleotide sequence homology of CV-B1 or other enterovirus serotype is higher.
(4) nucleotide sequence that above comparison is determined is carried out splicing and be organized into complete whole genome sequence.
The part VP1 gene order (being scribed ss primer) of table 5:E-33
Order-checking used by table 6E-33 whole genome amplification and amplimer
Remarks :-: position is uncertain
These are only some embodiments of the present invention, as long as employ the above technical scheme, all should protection scope of the present invention be fallen into.
Claims (1)
1. an amplification method for different enterovirus serotype full-length genome, is characterized in that, said method comprising the steps of:
(1) viral RNA is extracted, the VP1 district of enterovirus universal primer 222-224 to viral RNA is utilized to carry out RT-PCR amplification, then electrophoresis detection is carried out with sepharose, the voltage 120V of 1%, detected result with predict the outcome close, amplification gained Nucleotide object band is checked order, and carry out NCBIBLAST comparison, if enterovirus Reference Strains nucleotide sequence homology is greater than 75% in this inquiry sequence and GenBank database, namely think same serotype, namely obtain the nucleotide sequence of described virus VP 1 district gene simultaneously;
(2) conservative property of enterovirus 5 ' UTR and 3 ' UTR is utilized, a head end upstream primer EV1F(5 ' TTAAAACAGCCTGTGGGTTG3 ' is designed for enterovirus full-length genome) and a tail end downstream primer EV8R(5 ' CACCGAATGCGGAGAATTTA3 '), VP1 district actual nucleotide sequence viral according to step (1) amplification gained again, design the pairing upstream primer EV3F of a pairing downstream primer EV2R and described tail end downstream primer EV8R of a described head end upstream primer EV1F, then RT-PCR amplification is carried out with EV1F-EV2R and EV3F-EV8R respectively, its product with 1% sepharose, 120V carries out electrophoresis detection, if ultraviolet gel imaging instrument detected result has the electrophoretic band close to 2400bp with 5000bp namely to conform to expected results, amplification terminates, and check order with corresponding amplimer,
(3) check order, splice and arrange and check order, until obtain whole genome nucleotide sequence according to step (2) gained nucleotide sequence bamboo product sequencing primer.
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| CN112708698A (en) * | 2020-12-25 | 2021-04-27 | 中山大学 | Primer group for determining CVB3 virus whole gene sequence |
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