CN112359147A - Gene copy number absolute quantitative detection method based on Zika virus envelope gene - Google Patents
Gene copy number absolute quantitative detection method based on Zika virus envelope gene Download PDFInfo
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Abstract
The invention relates to an absolute quantitative detection method for gene copy number based on Zika virus envelope genes, belonging to the technical field of genes. The method comprises the steps of constructing a ZIKV E standard substance plasmid, designing a fluorescent quantitative PCR specific primer by referring to an obtained ZIKV E target gene sequence, and detecting the ZIKV E gene copy number in culture supernatant by using the obtained ZIKV E standard substance plasmid. The invention aims at ZIKV E gene construction standard substance plasmid, namely, the ZIKV E gene is constructed on a carrier in full length, a ZIKV fluorescence quantitative PCR absolute quantitative system is established, the detection system is established by detecting the ZIKV E gene, the ZIKV infection is rapidly detected, the ZIKV E gene copy number in the supernatant is absolutely quantified, and the ZIKV prevention and control technology is further supported.
Description
Technical Field
The invention belongs to the technical field of genes, and particularly relates to an absolute quantitative detection method for gene copy number based on Zika virus envelope genes.
Background
Zika virus (ZIKV) belongs to the flaviviridae family of flaviviridae, and is an arbovirus transmitted by aedes aegypti (a.aegypti) and aedes albopictus (a.albopictus). Zika virus infection can cause nervous system diseases such as fetal Microcephaly (Microcephaly) and severe brain malformation, and infected adults can cause Guillain-Barre syndrome (Guillain-Barr é syndrome), which leads to encephalitis, myelitis, and conjunctivitis, uveitis, etc., which are associated with ocular infections. The zika virus genome is a single-stranded positive-strand RNA, contains about 11000 nucleotides, contains one Open Reading Frame (ORF), encodes 3 structural proteins [ Capsid protein (Capsid, C), pre-membrane (PrM) protein, Envelope protein (Envelope, E) ] and 7 non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, NS 5). Zika virus can be classified into Asian and African based on genomic evolution analysis.
According to WHO reports that 84 countries and regions around the world have found the arbovirus transmission of the Zika virus, 13 regions report the interpersonal transmission of the Zika virus, and 60 countries and regions around the world have responded to the Zika virus. The WHO has announced the Zika virus as a global "public health emergency (public health emergency)". At present, input Zika case reports exist in Beijing, Guangdong, Zhejiang and the like, China has a high risk of input disease cases, and a good detection means is favorable for preventing and controlling Zika viruses. Currently, the laboratory detection method of ZIKV mainly includes virus isolation culture, serology, nucleic acid and biosensor detection, etc. The virus isolation and culture requirements are high, the time consumption is long, cross reaction exists among members in the flaviviridae family in the serology aspect, and the sensitivity of the emerging biosensor technology in detecting Zika virus needs to be improved.
Therefore, how to overcome the defects of the prior art is a problem which needs to be solved in the technical field at present.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides a Zika virus envelope gene-based absolute quantitative detection method for non-therapeutic and non-diagnostic purposes, which can quickly realize the absolute quantification of the copy number of the Zika virus envelope gene and provide technical support for ZIKV prevention and control.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
an absolute quantitative detection method based on the gene copy number of Zika virus envelope genes comprises the following steps:
(1) construction of ZIKV standard quality grains:
a. designing PCR forward and reverse specific primers of ZIKV E:
ZIKV E For：5'-atcaggtgcataggagtcag-3'；
ZIKV E Rev：5'-agcagagacggctgtggataag-3'；
cell culture of zikv: inoculating the Zika virus liquid on Vero cells, culturing until Vero cell lesions reach more than 80%, collecting culture supernatant, centrifuging and taking the supernatant to obtain virus liquid;
c. obtaining of positive strains: extracting ZIKV genomic RNA from a virus solution, carrying out reverse transcription to obtain cDNA, carrying out amplification PCR by using the primer pair in the step (1) a, connecting an amplification product to a pMD-19T Vector cloning Vector, transforming Top10 competent cells, selecting a single clone to carry out sequencing identification, and obtaining a positive strain with a ZIKV E target gene sequence identification accuracy;
d. copy number calculation of standard plasmid: transferring the positive strain with the correct sequence identification obtained in the step (1) c into an ampicillin-resistant LB liquid culture medium, carrying out shake culture in a shaking table at 37 ℃ for overnight, then extracting plasmids to obtain ZIKV standard plasmid, measuring the concentration of the ZIKV standard plasmid by using an ultraviolet spectrophotometer, and calculating the copy number of the standard plasmid according to the following formula:
Number of copies(copies/μl)＝(Amount×6.022×1023)/(Length×1×109×650)
wherein, Amount is the content of standard plasmid measured by an ultraviolet spectrophotometer, and the unit ng/mul; length represents the Length of template DNA;
the LB liquid culture medium with ampicillin resistance is LB liquid culture medium +100 mug/mL ampicillin;
(2) the following fluorescent quantitative PCR specific primers were designed:
ZIKV E-1F：5'-gacagtcacagtggaggtacag-3'；
ZIKV E-1R：5'-cgactcctatgacaatgtaaga-3'；
(3) and (3) detecting the ZIKV E gene copy number in the culture supernatant to be detected by using the ZIKV standard quality particles obtained in the step (1), wherein the specific detection method comprises the following steps:
a. diluting the ZIKV standard substance plasmid obtained in the step (1) by double distilled water according to gradient, obtaining an amplification curve and a dissolution curve of the ZIKV standard substance plasmid by adopting the primer real-time fluorescent quantitative PCR detection in the step (2) through an SYBR method, and taking the Ct value of the standard substance obtained by the fluorescent quantitative PCR detection as an X axis and the copy number log of the standard substance as an X axis10Drawing a standard curve of the ZIKV standard plasmid as a Y axis to obtain a standard curve equation of the ZIKV standard plasmid;
b. and (4) adopting the same PCR reaction system in the step (3) a, simultaneously detecting the ZIKV E gene copy number in the culture supernatant to be detected by SYBR real-time fluorescence quantitative PCR, obtaining a standard curve equation by using the standard plasmid according to the step (3) a, and further obtaining the ZIKV E gene copy number in the PCR detection culture supernatant.
Further, it is preferable that the specific method of ZIKV cell culture is: the Zika virus solution was inoculated into Vero cells washed with serum-free MEM solution at 37 ℃ with CO2Adsorbing for 1 hour at constant temperature in an environment with volume concentration of 5%, then supplementing a virus maintenance solution, culturing in the same environment for 4-5 days, when Vero cytopathic effect reaches more than 80%, collecting culture supernatant, centrifuging at 4 ℃ and 12000 Xg for 30min, and harvesting the supernatant to obtain virus solution, namely freezing and storing at-70 ℃;
the volume ratio of the Zika virus solution to the virus maintenance solution was 1: 9;
the virus maintenance liquid is MEM culture medium +2% (w/v) fetal bovine serum.
Further, it is preferable that the Vero cells are washed 2 times in serum-free MEM.
Further, it is preferable that the specific method for obtaining the positive strain is: extracting ZIKV genomic RNA from a virus solution, carrying out reverse transcription on the ZIKV genomic RNA solution by using a random primer to obtain cDNA, amplifying a ZIKV E gene by using the specific primer in the step (1) a through a PCR (polymerase chain reaction) method to obtain a corresponding amplification product, connecting the amplification product to a pMD-19T Vector cloning Vector, transforming Top10 competent cells, selecting a single clone, carrying out sequencing identification, and obtaining a positive strain with a ZIKV E target gene sequence identification accuracy.
Further, it is preferable that the rotation speed of shaking culture in the shaker in the step (1) d is 220 rpm.
Further, it is preferable that, in the step (3) a, the ZIKV standard plasmid obtained in the step (1) d is diluted to 10 degrees by gradient with double distilled water-9And then, obtaining an amplification curve and a dissolution curve of the ZIKV standard plasmid by SYBR real-time fluorescent quantitative PCR detection.
Further, it is preferable to dilute the solution to 10 degrees centigrade in a gradient with double distilled water-9The method is characterized in that the ZIKV standard substance plasmid is diluted into the following gradient by double distilled water: 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9。
Further, it is preferable that, in the step (3) a, the PCR amplification system is as shown in Table 1;
TABLE 1
| Composition (I) | Volume of | |
| Form panel | 1μl | |
| ZIKV E-1F，10μM | 1μl | |
| ZIKV E-1R， | 1μl | |
| 2×SYBR green Master Mix | 10μl | |
| Double distilled water | 7μl | |
| Total volume | 20μl |
Wherein the template is cDNA obtained by reverse transcription of extracted RNA;
the reaction procedure used was as follows:
(1)95℃3min；
(2)40 cycles: 10s at 95 ℃; 40s at 60 ℃; a read plate;
after the PCR reaction was completed, the temperature was gradually increased from 65 ℃ to 95 ℃ at 0.5 ℃/s to draw a dissolution curve.
Compared with the prior art, the invention has the beneficial effects that:
the invention aims at ZIKV E gene construction standard substance plasmid, namely, ZIKV E gene construction is carried out on a carrier, representative ZIKV Asia 13-strain and African 7-strain virus genomes are selected for sequence comparison analysis, conservative fluorescent quantitative PCR primers are designed, a ZIKV fluorescent quantitative PCR absolute quantitative system is established, the detection method can be used for carrying out absolute quantification on the gene copy number of Zika virus envelope genes, and technical support is provided for ZIKV prevention and control.
Drawings
FIG. 1 is a partial sequence alignment of ZIKV E genes of different strains;
FIG. 2 is a ZIKV standard quality particle electrophoretogram; in the figure, M: marker DL 5000;
FIG. 3 is a ZIKV standard plasmid and ZIKV sample amplification curve directly obtained from the fluorescent quantitative PCR assay of the examples;
FIG. 4 is a melting curve of the ZIKV standard plasmid and the ZIKV sample directly obtained by the fluorescent quantitative PCR assay of the example;
FIG. 5 shows the melting peaks of the ZIKV standard plasmid and the ZIKV sample directly obtained by the fluorescent quantitative PCR assay of the examples;
FIG. 6 is a calibration curve of the E gene region of the plasmid of the ZIKV standard in the example.
Detailed Description
The present invention will be described in further detail with reference to examples.
It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The materials or equipment used are not indicated by manufacturers, and all are conventional products available by purchase.
Example 1
An absolute quantitative detection method based on the gene copy number of Zika virus envelope genes comprises the following steps:
(1) construction of ZIKV standard quality grains:
a. designing PCR forward and reverse specific primers of ZIKV E:
ZIKV E For：5'-atcaggtgcataggagtcag-3'；
ZIKV E Rev：5'-agcagagacggctgtggataag-3'；
cell culture of zikv: inoculating the Zika virus liquid on Vero cells, culturing until Vero cell lesions reach more than 80%, collecting culture supernatant, centrifuging and taking the supernatant to obtain virus liquid;
c. obtaining of positive strains: extracting ZIKV genomic RNA from a virus solution, carrying out reverse transcription to obtain cDNA, carrying out amplification PCR by using the primer pair in the step (1) a, connecting an amplification product to a pMD-19T Vector cloning Vector, transforming Top10 competent cells, selecting a single clone to carry out sequencing identification, and obtaining a positive strain with a ZIKV E target gene sequence identification accuracy;
d. copy number calculation of standard plasmid: transferring the positive strain with the correct sequence identification obtained in the step (1) c into an ampicillin-resistant LB liquid culture medium, carrying out shake culture in a shaking table at 37 ℃ for overnight, then extracting plasmids to obtain ZIKV standard plasmid, measuring the concentration of the ZIKV standard plasmid by using an ultraviolet spectrophotometer, and calculating the copy number of the standard plasmid according to the following formula:
Number of copies(copies/μl)＝(Amount×6.022×1023)/(Length×1×109×650)
wherein, Amount is the content of standard plasmid measured by an ultraviolet spectrophotometer, and the unit ng/mul; length represents the Length of template DNA;
the LB liquid culture medium with ampicillin resistance is LB liquid culture medium +100 mug/mL ampicillin;
(2) the following fluorescent quantitative PCR specific primers were designed:
ZIKV E-1F：5'-gacagtcacagtggaggtacag-3'；
ZIKV E-1R：5'-cgactcctatgacaatgtaaga-3'；
(3) and (3) detecting the ZIKV E gene copy number in the culture supernatant to be detected by using the ZIKV standard quality particles obtained in the step (1), wherein the specific detection method comprises the following steps:
a. diluting the ZIKV standard substance plasmid obtained in the step (1) by double distilled water according to gradient, obtaining an amplification curve and a dissolution curve of the ZIKV standard substance plasmid by adopting the primer real-time fluorescent quantitative PCR detection in the step (2) through an SYBR method, and taking the Ct value of the standard substance obtained by the fluorescent quantitative PCR detection as an X axis and the copy number log of the standard substance as an X axis10Drawing a standard curve of the ZIKV standard plasmid as a Y axis to obtain a standard curve equation of the ZIKV standard plasmid;
b. and (4) adopting the same PCR reaction system in the step (3) a, simultaneously detecting the ZIKV E gene copy number in the culture supernatant to be detected by SYBR real-time fluorescence quantitative PCR, obtaining a standard curve equation by using the standard plasmid according to the step (3) a, and further obtaining the ZIKV E gene copy number in the PCR detection culture supernatant.
Example 2
An absolute quantitative detection method based on the gene copy number of Zika virus envelope genes comprises the following steps:
(1) construction of ZIKV standard quality grains:
a. designing PCR forward and reverse specific primers of ZIKV E:
ZIKV E For：5'-atcaggtgcataggagtcag-3'；
ZIKV E Rev：5'-agcagagacggctgtggataag-3'；
cell culture of zikv: inoculating the Zika virus liquid on Vero cells, culturing until Vero cell lesions reach more than 80%, collecting culture supernatant, centrifuging and taking the supernatant to obtain virus liquid;
c. obtaining of positive strains: extracting ZIKV genomic RNA from a virus solution, carrying out reverse transcription to obtain cDNA, carrying out amplification PCR by using the primer pair in the step (1) a, connecting an amplification product to a pMD-19T Vector cloning Vector, transforming Top10 competent cells, selecting a single clone to carry out sequencing identification, and obtaining a positive strain with a ZIKV E target gene sequence identification accuracy;
d. copy number calculation of standard plasmid: transferring the positive strain with the correct sequence identification obtained in the step (1) c into an ampicillin-resistant LB liquid culture medium, carrying out shake culture in a shaking table at 37 ℃ for overnight, then extracting plasmids to obtain ZIKV standard plasmid, measuring the concentration of the ZIKV standard plasmid by using an ultraviolet spectrophotometer, and calculating the copy number of the standard plasmid according to the following formula:
Number of copies(copies/μl)＝(Amount×6.022×1023)/(Length×1×109×650)
wherein, Amount is the content of standard plasmid measured by an ultraviolet spectrophotometer, and the unit ng/mul; length represents the Length of template DNA;
the LB liquid culture medium with ampicillin resistance is LB liquid culture medium +100 mug/mL ampicillin;
(2) the following fluorescent quantitative PCR specific primers were designed:
ZIKV E-1F：5'-gacagtcacagtggaggtacag-3'；
ZIKV E-1R：5'-cgactcctatgacaatgtaaga-3'；
(3) and (3) detecting the ZIKV E gene copy number in the culture supernatant to be detected by using the ZIKV standard quality particles obtained in the step (1), wherein the specific detection method comprises the following steps:
a. double steaming the ZIKV standard substance plasmid obtained in the step (1)Diluting with water in a gradient manner, obtaining an amplification curve and a dissolution curve of the ZIK V standard substance plasmid by adopting the primer real-time fluorescent quantitative PCR detection in the step (2) through an SYBR method, and taking the Ct value of the standard substance obtained by the fluorescent quantitative PCR detection as an X axis and the copy number log of the standard substance10Drawing a standard curve of the ZIKV standard plasmid as a Y axis to obtain a standard curve equation of the ZIKV standard plasmid;
b. and (4) adopting the same PCR reaction system in the step (3) a, simultaneously detecting the ZIKV E gene copy number in the culture supernatant to be detected by SYBR real-time fluorescence quantitative PCR, obtaining a standard curve equation by using the standard plasmid according to the step (3) a, and further obtaining the ZIKV E gene copy number in the PCR detection culture supernatant.
The specific method for culturing the ZIKV cells comprises the following steps: the Zika virus solution was inoculated into Vero cells washed with serum-free MEM solution at 37 ℃ with CO2Adsorbing for 1 hour at constant temperature in an environment with volume concentration of 5%, then supplementing a virus maintenance solution, culturing in the same environment for 4-5 days, when Vero cytopathic effect reaches more than 80%, collecting culture supernatant, centrifuging at 4 ℃ and 12000 Xg for 30min, and harvesting the supernatant to obtain virus solution, namely freezing and storing at-70 ℃;
the volume ratio of the Zika virus solution to the virus maintenance solution was 1: 9;
the virus maintenance liquid is MEM culture medium +2% (w/v) fetal bovine serum.
The Vero cells were washed 2 times in serum-free MEM.
The specific method for obtaining the positive strain comprises the following steps: extracting ZIKV genomic RNA from a virus solution, carrying out reverse transcription on the ZIKV genomic RNA solution by using a random primer to obtain cDNA, then amplifying a ZIK V E gene by using the specific primer in the step (1) a through a PCR (polymerase chain reaction) method to obtain a corresponding amplification product, connecting the amplification product to a pMD-19T Ve vector cloning vector, transforming Top10 competent cells, selecting a single clone for sequencing identification, and obtaining a positive strain with a correct ZIKV E target gene sequence identification.
In the step (1) d, the rotation speed of shaking culture in a shaking table is 220 rpm.
In the step (3) a, the ZIKV standard substance obtained in the step (1) dThe plasmid was diluted to 10 degrees in double distilled water-9And then, obtaining an amplification curve and a dissolution curve of the ZIK V standard plasmid by SYBR real-time fluorescent quantitative PCR detection.
Diluting with double distilled water to 10% by gradient-9The method is characterized in that the ZIKV standard substance plasmid is diluted into the following gradient by double distilled water: 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9。
In the step (3) a, a PCR amplification system is shown in Table 1;
wherein the template is cDNA obtained by reverse transcription of extracted RNA;
the reaction procedure used was as follows:
(1)95℃3min；
(2)40 cycles: 10s at 95 ℃; 40s at 60 ℃; a read plate;
after the PCR reaction was completed, the temperature was gradually increased from 65 ℃ to 95 ℃ at 0.5 ℃/s to draw a dissolution curve.
Examples of the applications
The serum-free MEM solution in the invention is sold as MEM/EBSS Hyclone.
(1) Constructing a ZIKV standard plasmid:
a. specific primers in Table 2 were designed for the E gene region based on the ZIKV (GenBank: MF036115) gene sequence in NCBI database:
PCR forward and reverse primers for ZIKV E:
ZIKV E For：5'-atcaggtgcataggagtcag-3'；(SEQ ID NO.1)
ZIKV E Rev：5'-agcagagacggctgtggataag-3'；(SEQ ID NO.2)
the sequences of HPV type 16E 6/E7 in the NCBI database (NCBI Reference Sequ: NC-001526.2), specific primers were designed according to Table 2 to amplify the E6/E7 region gene sequences:
TABLE 2 PCR primers for ZIKV E gene
Cell culture of zikv: inoculating 1ml Zika virus solution to Vero cells which have been densely grown and good in transparency and washed for 2 times by serum-free MEM solution, and firstly, inoculating 1ml of Zika virus solution to Vero cells which have been washed by CO solution at 37 ℃2Adsorbing at constant temperature in 5% volume for 1 hr, and adding 9ml virus maintaining solution [ MEM medium +2% (w/v) fetal calf serum]Culturing in the same environment for 4-5 days, collecting culture supernatant when Vero cytopathic effect (CPE) reaches above 80%, centrifuging at 4 deg.C and 12000 Xg for 30min, and harvesting supernatant to obtain virus solution, and freezing at-70 deg.C.
c. Extracting ZIKV genomic RNA from 140 μ l of virus solution, dissolving in 60 μ l of nuclease-free water, collecting 8 μ l of ZIKV genomic RNA solution, and purifying with random primer pd (N)6Performing reverse transcription to obtain cDNA, amplifying a ZIKV E gene by using a specific primer in the step (1) a through a PCR method to obtain a corresponding amplification product, wherein the gene sequence of the amplification primer is shown as SEQ ID NO.3, connecting the amplification product to a pMD-19T Vec or cloning vector, transforming Top10 competent cells, selecting a single clone for sequencing identification, and obtaining a positive strain with a correct ZIKV target gene sequence identification;
d. transferring the positive strain with the correct sequence identification obtained in the step (1) c into 5mL of LB liquid culture medium with ampicillin resistance, carrying out shake culture in a shaking table at the temperature of 37 ℃ and the rotating speed of 220rpm overnight, then extracting plasmids to obtain ZIKV standard substance plasmids, measuring the concentration of the ZIKV standard substance plasmids by using an ultraviolet spectrophotometer, and calculating the copy number of the standard substance plasmids according to the following formula:
Number of copies(copies/μl)＝(Amount×6.022×1023)/(Length×1×109×650)
wherein, Amount is the content (ng/mu l) of standard plasmid measured by an ultraviolet spectrophotometer, and Length represents the Length of template DNA.
The Length of the ZIKV standard plasmid DNA is 4204 bp. In this example, the ZIKV standard quality particle Amount was measured to be 197.3 ng/. mu.l by an ultraviolet spectrophotometer, and the calculated ZIKV standard quality particle copy number was 4.35X 1010copies/μl。
(2) Aligning the gene sequences of ZIKV 13 Asia (MF036115, KU497555, MT483911, KU681081, JN860885, KJ776791, EU545988, MK269360, KF993678, MK829154, KY606271, MH063264, HQ234499) and 7 Africa (NC-012532, HQ234500, HQ234501, KF268948, KF383115, KF383116, KF383118) in NCBI database to obtain the conserved region of E gene, designing the specific primers for fluorescent quantitative PCR in Table 3 with reference to the target gene sequence of ZIKV obtained in step (1):
TABLE 3 ZIKV E fluorescent quantitative PCR primers
The gene sequence of the amplification product is shown as SEQ ID NO. 6.
(3) And (3) utilizing the ZIKVE gene copy number in the culture supernatant of the ZIKV standard quality particles obtained in the step (1), wherein the specific detection method comprises the following steps:
a. diluting the ZIKV standard substance plasmid obtained in the step (1) d to 10 degrees by double distilled water according to gradient-9I.e. the following gradient: 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9Using the fluorescent quantitative PCR primers in Table 3, obtaining an amplification curve (shown in figure 3), a dissolution curve (shown in figure 4) and a dissolution peak (shown in figure 5) of the ZIKV standard plasmid through SYBR real-time fluorescent quantitative PCR detection according to a reaction system in Table 4, drawing a standard curve (shown in figure 6) of the ZIKV standard plasmid by taking a Ct value (shown in figure 5) of the standard obtained through the fluorescent quantitative PCR detection as an X axis and taking a copy number log10 (shown in figure 5) of the standard as a Y axis, and further obtaining a standard curve equation of the ZIKV standard plasmid: y-0.328 x +12.816 (R)2＝0.99497)。
The PCR reaction system is shown in Table 4:
TABLE 4 fluorescent quantitative PCR reaction system (total volume of reaction 20. mu.l)
| Composition (I) | Volume of |
| Form panel | 1μl |
| ZIKV E-1F，10μM | 1μl |
| ZIKV E-1R，10μM | 1μl |
| 2×SYBR green Master Mix | 10μl |
| Double distilled water | 7μl |
| Total volume | 20μl |
The reaction procedure used was as follows:
(1)95℃3min；
(2)40 cycles: 10s at 95 ℃; 40s at 60 ℃; a read plate;
immediately after the PCR reaction was completed, the temperature was gradually increased from 65 ℃ to 95 ℃ at 0.5 ℃/s to draw a dissolution curve.
TABLE 5 ZIKV Standard E Gene test results
b. And (3) detecting the ZIKV E gene copy number in 8 parts of culture supernatant to be detected by a SYBR method real-time fluorescence quantitative PCR according to the PCR reaction system in the step (3) a, wherein a template is cDNA obtained by reverse transcription (the reaction volume is 20 mul) of 8 mul of RNA solution, obtaining a ZIKV amplification curve (shown in figure 3), a dissolving curve (shown in figure 4) and a dissolving peak (shown in figure 5) in a sample, and obtaining the ZIKV E gene copy number in the culture supernatant to be detected (shown in figure 6) according to a standard curve equation obtained by the step (3) a by using standard plasmid.
TABLE 6 detection results of ZIKV E genes in culture supernatant samples
The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
<110> institute of medical science and biology of China academy of medical sciences
<120> absolute quantitative determination method for gene copy number based on Zika virus envelope gene
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atcaggtgca taggagtcag caatagggac tttgtggaag gtatgtcagg tgggacttgg 60
gttgatgttg tcttggaaca tggaggttgt gtcaccgtaa tggcacagga caaaccgact 120
gtcgacatag agctggttac aacaacagtc agcaacatgg cggaggtaag atcctactgc 180
tatgaggcat caatatcgga catggcttcg gacagccgct gcccaacaca aggtgaagcc 240
taccttgaca agcaatcaga cactcaatat gtctgcaaaa gaacgttagt ggacagaggc 300
tggggaaatg gatgtggact ttttggcaaa gggagcctgg tgacatgcgc taagtttgca 360
tgctccaaga aaatgaccgg gaagagcatc cagccagaga atctggagta ccggataatg 420
ctgtcagttc atggctccca gcacagtggg atgatcgtta atgacacagg acatgaaact 480
gatgagaata gagcgaaggt tgagataacg cccaattcac caagagccga agccaccctg 540
gggggttttg gaagcctagg acttgattgt gaaccgagga caggccttga cttttcagat 600
ttgtattact tgactatgaa taacaagcac tggttggttc acaaggagtg gttccacgac 660
attccattac cttggcacgc tggggcagac accggaactc cacactggaa caacaaagaa 720
gcactggtag agttcaagga cgcacatgcc aaaaggcaaa ctgtcgtggt tctagggagt 780
caagaaggag cagttcacac ggcccttgct ggagctctgg aggctgagat ggatggtgca 840
aagggaaggc tgtcctctgg ccacttgaaa tgtcgcctga aaatggataa acttagattg 900
aagggcgtgt catactcctt gtgtaccgca gcgttcacat tcaccaagat cccggctgaa 960
acactgcacg ggacagtcac agtggaggta cagtacgcag ggacagatgg accttgcaag 1020
gttccagctc agatggcggt ggacatgcaa actctgaccc cagttgggag gctgataacc 1080
gctaaccccg taatcactga aagcactgag aactccaaga tgatgctgga acttgatcca 1140
ccatttgggg actcttacat tgtcatagga gtcggggaga agaagatcac ccaccactgg 1200
cacaggagtg gcagcaccat tggaaaagca tttgaagcca ctgtgagagg tgccaggaga 1260
atggcagtct tgggagacac agcctgggac tttggatcag ttggaggcgc tctcaactca 1320
ttgggcaagg gcatccatca aatttttgga gcagctttca aatcattgtt tggaggaatg 1380
tcctggttct cacaaattct cattggaacg ttgctgatgt ggttgggtct gaacacaaag 1440
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gacagtcaca gtggaggtac agtacgcagg gacagatgga ccttgcaagg ttccagctca 60
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aatcactgaa agcactgaga actccaagat gatgctggaa cttgatccac catttgggga 180
ctcttacatt gtcataggag tcg 203
Claims (8)
1. The absolute quantitative detection method based on the copy number of the gene of the envelope gene of Zika virus is characterized by comprising the following steps of:
(1) construction of ZIKV standard quality grains:
a. designing PCR forward and reverse specific primers of ZIKV E:
ZIKV E For：5'-atcaggtgcataggagtcag-3'；
ZIKV E Rev：5'-agcagagacggctgtggataag-3'；
b. cell culture of ZIKV: inoculating the Zika virus liquid on Vero cells, culturing until Vero cell lesions reach more than 80%, collecting culture supernatant, centrifuging and taking the supernatant to obtain virus liquid;
c. obtaining of positive strains: extracting ZIKV genomic RNA from a virus solution, carrying out reverse transcription to obtain cDNA, carrying out amplification PCR by using the primer pair in the step (1) a, connecting an amplification product to a pMD-19T Vector cloning Vector, transforming Top10 competent cells, selecting a single clone to carry out sequencing identification, and obtaining a positive strain with a ZIKV E target gene sequence identification accuracy;
d. copy number calculation of standard plasmid: transferring the positive strain with the correct sequence identification obtained in the step (1) c into an ampicillin-resistant LB liquid culture medium, carrying out shake culture in a shaking table at 37 ℃ for overnight, then extracting plasmids to obtain ZIKV standard plasmid, measuring the concentration of the ZIKV standard plasmid by using an ultraviolet spectrophotometer, and calculating the copy number of the standard plasmid according to the following formula:
Number of copies（copies/μl）=(Amount×6.022×1023)/（Length×1×109×650）
wherein, Amount is the content of standard plasmid measured by an ultraviolet spectrophotometer, and the unit ng/mul; length represents the Length of template DNA;
the LB liquid culture medium with ampicillin resistance is LB liquid culture medium +100 mug/mL ampicillin;
(2) the following fluorescent quantitative PCR specific primers were designed:
ZIKV E-1F：5'-gacagtcacagtggaggtacag-3'；
ZIKV E-1R：5'-cgactcctatgacaatgtaaga-3'；
(3) and (3) detecting the ZIKV E gene copy number in the culture supernatant to be detected by using the ZIKV standard quality particles obtained in the step (1), wherein the specific detection method comprises the following steps:
a. diluting the ZIKV standard substance plasmid obtained in the step (1) by double distilled water according to gradient, obtaining an amplification curve and a dissolution curve of the ZIKV standard substance plasmid by adopting the primer real-time fluorescent quantitative PCR detection in the step (2) through an SYBR method, and taking the Ct value of the standard substance obtained by the fluorescent quantitative PCR detection as an X axis and the copy number log of the standard substance as an X axis10Drawing a standard curve of the ZIKV standard plasmid as a Y axis to obtain a standard curve equation of the ZIKV standard plasmid;
b. and (4) adopting the same PCR reaction system in the step (3) a, simultaneously detecting the ZIKV E gene copy number in the culture supernatant to be detected by SYBR real-time fluorescence quantitative PCR, obtaining a standard curve equation by using the standard plasmid according to the step (3) a, and further obtaining the ZIKV E gene copy number in the PCR detection culture supernatant.
2. The method for the absolute quantitative determination of the copy number of genes based on the envelope gene of Zika virus according to claim 1, wherein the specific method for culturing the cells of ZIKV comprises: the Zika virus solution was inoculated into Vero cells washed with serum-free MEM solution at 37 ℃ with CO2Adsorbing at constant temperature for 1 hr in 5% environment, adding virus maintaining liquid, culturing in the same environment for 4-5 days, collecting culture supernatant when Vero cytopathic effect reaches 80% or more, and culturing at 4 deg.C and 1 deg.CCentrifuging at 2000 Xg for 30min, and collecting supernatant to obtain virus solution, and freezing at-70 deg.C;
the volume ratio of the Zika virus solution to the virus maintenance solution was 1: 9;
the virus maintenance liquid is MEM culture medium +2% (w/v) fetal bovine serum.
3. The method for the absolute quantitative determination of the copy number of a gene based on the envelope gene of Zika virus according to claim 2, wherein the Vero cells are washed 2 times in serum-free MEM.
4. The absolute quantitative determination method for gene copy number based on Zika virus envelope gene according to claim 1, characterized in that the specific method for obtaining the positive strain is: extracting ZIKV genomic RNA from a virus solution, carrying out reverse transcription on the ZIKV genomic RNA solution by using a random primer to obtain cDNA, amplifying a ZIKV E gene by using the specific primer in the step (1) a through a PCR (polymerase chain reaction) method to obtain a corresponding amplification product, connecting the amplification product to a pMD-19T Vector cloning Vector, transforming Top10 competent cells, selecting a single clone, carrying out sequencing identification, and obtaining a positive strain with a ZIKV E target gene sequence identification accuracy.
5. The method according to claim 1, wherein in step (1) d, the rotational speed during shaking culture in a shaker is 220 rpm.
6. The method for the absolute quantitative determination of the copy number of a gene based on the envelope gene of Zika virus according to claim 1, wherein in the step (3) a, the ZIKV standard plasmid obtained in the step (1) d is diluted to 10 degrees by gradient with double distilled water-9And then, obtaining an amplification curve and a dissolution curve of the ZIKV standard plasmid by SYBR real-time fluorescent quantitative PCR detection.
7. The gene copy number based on the envelope gene of Zika virus according to claim 6The method for absolute quantitative determination of (1), characterized in that it is diluted to 10 degrees by gradient with double distilled water-9The method is characterized in that the ZIKV standard substance plasmid is diluted into the following gradient by double distilled water: 10-1、10-2、10-3、10-4、10-5、10-6、10-7、10-8、10-9。
8. The method for the absolute quantitative determination of the copy number of a gene based on an envelope gene of Zika virus according to claim 1, wherein in the step (3) a, the PCR amplification system is shown in Table 1;
TABLE 1
Wherein the template is cDNA obtained by reverse transcription of extracted RNA;
the reaction procedure used was as follows:
（1）95℃ 3 min；
(2)40 cycles: 10s at 95 ℃; 40s at 60 ℃; a read plate;
after the PCR reaction was completed, the temperature was gradually increased from 65 ℃ to 95 ℃ at 0.5 ℃/s to draw a dissolution curve.
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