CN112239778A

CN112239778A - Hepatitis C virus detection kit

Info

Publication number: CN112239778A
Application number: CN201910643838.5A
Authority: CN
Inventors: 郑超; 吴祚达; 陈素华
Original assignee: Nky Bochang Wuhan Biotechnology Co ltd
Current assignee: Nky Bochang Wuhan Biotechnology Co ltd
Priority date: 2019-07-17
Filing date: 2019-07-17
Publication date: 2021-01-19

Abstract

The invention discloses a hepatitis C virus detection kit, wherein the kit comprises two pairs of amplification primers and a multiplex PCR mixed reaction solution, wherein the amplification primers are used for amplifying obesity gene polymorphic sites including an rs8099917 site and an rs12979860 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site. The invention adopts the hepatitis C virus detection kit and the multiplex PCR amplification method to simultaneously amplify two important SNP sites of IL-28B, can flexibly select the amplified site combination according to the actual requirements, can play a role of one box for multiple purposes, and has the advantages of high primer specificity, low mismatching rate, primer length and T_mThe value design is reasonable, and the amplification product is convenient to detect and separate. Multiple target products are amplified at one time, so that the amplification efficiency is greatly improved, and the amplification cost is reduced. The detection kit can be expanded to other SNP locus detection, and has good popularization value.

Description

Hepatitis C virus detection kit

Technical Field

The invention relates to a hepatitis C virus detection kit, and belongs to the field of detection kits.

Background

Viral Hepatitis C (HCV) is an important type of chronic hepatitis, is one of the most prevalent infectious diseases in the world at present, and causes Chronic Hepatitis C (CHC) which is hidden and easily ignored by patients, and is one of the main causes of cirrhosis, liver cancer and liver failure. The international standard scheme for treating CHC is polyethylene glycol interferon alpha (Peg-IFN alpha) combined with Ribavirin (RBV), and researches in recent years prove that the clinical curative effect of CHC antiviral treatment is highly related to interleukin-28B (IL-28B) single nucleotide polymorphism which is positioned on human chromosome 19 and codes interferon lambda 3 besides the influence of virus load, age, race and liver morphological factors, but most reports are concentrated on IL-28B rs8099917 site, and the predictive value of IL-28B on the curative effect and prognosis of hepatitis C treatment is still in the initial research stage.

However, in addition to the IL-28B rs8099917 site, the IL-28B rs12979860 site has been reported to be associated with susceptibility to hepatitis B virus. Because hepatitis c is prevalent, the development of a rapid detection kit for hepatitis c and its prognosis is clinically significant.

Disclosure of Invention

In view of the above problems in the prior art, the present invention aims to obtain a hepatitis c virus detection kit.

In order to achieve the purpose, the technical scheme of the hepatitis C virus detection kit adopted by the invention is as follows:

the kit comprises two pairs of amplification primers and a multiplex PCR mixed reaction solution, wherein the amplification primers are used for amplifying obesity gene polymorphism sites including an rs8099917 site and an rs12979860 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site.

Preferably, the sequence of the amplification primer of the rs8099917 site is shown as a sequence table SEQ ID NO: 1 to 2.

Preferably, the amplification primer sequence of the rs12979860 locus is shown in a sequence table SEQ ID NO: 3 to 4.

Preferably, the mixed reaction solution for multiplex PCR comprises DNA polymerase and Mg²⁺And dNTPs. More preferably, the multiplex PCR mixture further comprises a PCR stabilizer and an enhancer.

Preferably, in the amplification system, the addition amount of the amplification primer and the template at each SNP site is the same.

The detection kit generally adopts a treated DNA sample as an amplification template, the sample is generally derived from human whole blood or tissues, and the interference of immunoglobulin G, hemoglobin and lactoferrin in the blood on a PCR reaction system can be reduced by treating the sample.

Preferably, the detection kit further comprises a whole blood sample PCR buffer solution, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has pH of 9.3-9.5. Whole blood sample PCR buffer is a preferred embodiment of the present invention, and other types of whole blood PCR reagents can be used without limitation to the buffer and buffer formulations of the present invention.

Preferably, the kit amplification system is a 50 μ L PCR system, and the system simultaneously contains amplification primers of rs8099917 site and rs12979860 site.

The amplification system is specifically as follows:

2×Platinum Multiplex PCR Master Mix 25μL；

2 mu L of template;

upstream and downstream primers (10. mu. mol/L) of rs8099917 and rs12979860 respectively are 1. mu.L; sterile double distilled water is filled to 50 mu L.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 10 min; then denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 90s, and extension at 72 ℃ for 60s, after 40 cycles, extension at 72 ℃ is carried out again for 10min, and the temperature is restored to 4 ℃.

The kit generally adopts a purified DNA sample for amplification detection. However, the kit also comprises a whole blood amplification buffer solution, so that the collected human whole blood sample can be directly amplified.

The invention also aims to provide a detection method of the hepatitis C virus detection kit, the detection kit adopts a multiplex PCR detection method to simultaneously amplify the rs8099917 site and the rs12979860 site, and an amplified product is detected after amplification.

The third purpose of the invention is to provide the application of the hepatitis C virus detection kit, and the application of the detection kit in the detection of the hepatitis C morbidity and/or prognosis.

Compared with the prior art, the invention adopts the hepatitis C virus detection kit and the multiplex PCR amplification method to simultaneously amplify two IL-28B genesImportant SNP sites, and can flexibly select amplified site combinations according to actual needs, can play a role of one box for multiple purposes, and the primer in the kit has high specificity, low mismatching rate, primer length and T_mThe value design is reasonable, and the amplification product is convenient to detect and separate. Multiple target products are amplified at one time, so that the amplification efficiency is greatly improved, and the amplification cost is reduced. The detection kit can be expanded to other SNP locus detection, and has good popularization value.

Detailed Description

The hepatitis C virus detection kit provided by the present invention will be described in detail and fully below with reference to examples. The following examples are illustrative only and are not to be construed as limiting the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.

First, design primer

Primer design is carried out according to polymorphic sites published in NCBI, and rs8099917 site is G/T base mutation and is expressed by K. The specific sequence is as follows:

CTGTGTCTTG CTTTCTCTTT CTCTCTCTCT CTCTGTTCCT GTCTCTGTCT CTGGCGTGAC TCCATCTTTC TTATTCATTT TTCCAACAAG CATCCTGCCC CAGGTCGCTC TGTCTGTCTC AATCAATCTC TTTTTGTTTA AACATAAAAT CTACTTCTTT TTCTGATAAA ATTAGCCATG CATGTTCAGT GGATAAAGCT TGAAAAATAC ACAAAAACAT AAAAGAAAAA AAACATCACC TATAACTTCA CCATCCTCCT CTCATCCCTC ATCCCACTTC TGGAACAAAT CGTCCCAATA CATAGGAATT TTCCATGTGT TTATTTGTGC ATATGTTTTC TGACTACCAA AGTAACACTT GTTCCTTGTA AAAGATTCCA TCCATACAAA AACATACAAC ATGGAGAGTT AAAGTAAGTC TTGTATTTCA CCTCCTGGAG GTAAATATTT TTTAACAATT TGTCACTGTT CCTCCTTTTG TTTTCCTTTC TGTGAGCAAT

K

TCACCCAAAT TGGAACCATG CTGTATACAG TTTGGTAGCT GGCTTTTTAT GTCTTACCAT TATCTCTCAT TTGCATTCTC CCACATCTTT AATTATAGCG TATCAGTTAG GGCCCAGCAG GAAACAGATG GCCCGTCAAT TTAGGATAAT TTGAGGTGGG GTTGATACCA GAAGCCTTTA TAAAGCGATT GGATAGTAAA GGCAAATGAC AAGAGCTAGT GCAAGCTCCT GGGGCCAGCA TGGGTGAGGG GCCTCATCCA CAGGCCTAAA GGAAGGGGAG AGGGTTGAGG GTGTGCATGT GGTTGCCTGA CTTGGGGAGG AGAGAGTCAC TTAGATTTGT GGAGGAAACA GATGAATTGT GGTGGCCCAG CACAGGGAGG AGCCCTGGGG AAAAAACACC TGACCTCCTC TCCCCTCTAC CTGGGGCCCA GAGTAGGTTG GAGAAGCAGG GACAGTAGAT AAGGAGGGGC AAACGGAAGA CATCCCCCAC CCCCACACCA

primer design is carried out according to polymorphic sites published in NCBI, and the rs12979860 site is a 'C/T' base mutation and is indicated by 'Y'. The specific sequence is as follows:

GGCCTGAGGA TGCAGAGAAG CTGGCGGGGG AGAGGGGCGG CGGGGCGCGG CCGTCACTCA CGCAGGCCGC CACATCCCTC CCCGCGGCCG CCAGCAGCTC CAGGATCGGG CCGGCGCCGG GGAGCAGCTC CGAGCGGTGC AGGCCGCTGA GCACTGCCTG GGCGTCCGCG ATGCCCCGGG CCACGTGGCG GAGCCGAGCG CAGGACTGCG GGGACGAGAG GGCGTTAGAG CGGGCCGCGC CCGGGCCATG CCTCTCCCGC CCACTCCCGG GCCTCACCGA TGGCCGCGGA GGATCCCTCC TGGGGCGGAA GGAGCAGTTG CGCTGCCCCC AGCTCAGCGC CTCTTCCTCC TGCGGGACAA GCGGCGCTTA TCGCATACGG CTAGGCCCCC TCGCCAGGGC CCCTAACCTC TGCACAGTCT GGGATTCCTG GACGTGGATG GGTACTGGCA GCGCACGGTC GTGCCTGTCG TGTACTGAAC CAGGGAGCTC CCCGAAGGCG

Y

GAACCAGGGT TGAATTGCAC TCCGCGCTCC CCCAGCAAAG CCCCTCGCCC CGACCTGGAG CCGAGTCCTC CCGGCAGGGC TCCCTTCTGT GATTGACCCT GAGCCTGCGT TCGCGCTGAC GACGGGGACT GCGGGGGTCT CGTGGTGGGA ATTGTGGGCG CTGACATAGG AGAGGCGCCT GCTGGGCGCT AGGACGCAGG ACCCCTTGGG ACAGGAACGG GTGTATGGGA ACCCGGTGGG GCCAGGGTCC CAGGGGGCAC AGGGGCTGGG CGGTGACTTA CGTAGCGGTC CCTCAGCGCC TTGGCAGCCG CCAGCGTCCG GGGCTCCAGC GAGCGGTAGT GCGAGAGCAG GCAGCGCCGG GGGGCCTTCT GCGATCACCG TGCACAGGAC CCACAGCCCC GCGGCCACTG CGGCCCAGAC ACTCGGCCGC ATCTCTGCTT CTGCAGCAGG CGAGAGACGT CAGGGAAGCC AAAGAGAGGG TCCAGCGCGT CCAGCCCCCC

specific amplification primers are designed according to the sequences, so that rapid and efficient amplification is facilitated. The primer sequences are as follows:

in this example, a multiplex PCR method was used to simultaneously amplify two SNP sites and then detect them.

II, kit composition

The rapid detection kit in this embodiment is a multiplex PCR kit for simultaneously amplifying two higher SNP sites of IL-28B, including upstream and downstream amplification primers and amplification template extraction reagents for two pairs of SNP sites, and a multiplex PCR mixed reaction solution, where the reaction solution includes DNA polymerase, Mg²⁺dNTPs, PCR stabilizers and enhancers.

The DNA template used in the kit of this embodiment is a human blood DNA template extracted after processing, and the collected human whole blood or tissue needs to be processed in advance. If the sample to be amplified is an unprocessed whole blood sample, a buffer solution for PCR of the whole blood sample is adopted, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has the pH value of 9.3-9.5.

Triple, multiplex PCR

The Multiplex PCR selects Platinum Multiplex PCR Master Mix as a reaction system, which comprises DNA polymerase and Mg²⁺dNTPs, PCR stabilizer and enhancer, wherein the Invitrogen Platinum Multiplex PCR Master Mix is selected in the experiment, and the PCR system is 50 mu L, wherein

2×Platinum Multiplex PCR Master Mix 25μL；

2 mu L of template;

Fourth, detection of amplification result

1. Amplification product detection

Because multiple PCR has more influence factors, reaction conditions, primer sites, amplification products and other information need to be comprehensively considered during amplification, and the amplification primer combination in the embodiment is repeatedly verified through multiple experiments, and the multiple PCR can be realized with the minimum mutual influence.

After the upstream and downstream primers in the embodiment are used for combined amplification, amplification products are verified, 2% agarose gel electrophoresis is selected for PCR products, bands 1 and 2 in the electrophoresis result are the amplification product sequence of rs12979860, bands 3 and 4 are the amplification product sequence of rs17817449, and the position of the electrophoresis band indicates that the amplification result is correct.

And recovering the electrophoresis result gel and then sequencing, wherein the sequencing result is consistent with the sequence of the target product.

2. SNP locus genotype frequency of IL-28B gene

Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims

1. A hepatitis C virus detection kit is characterized by comprising two pairs of amplification primers and a multiplex PCR mixed reaction solution, wherein the amplification primers are used for amplifying obesity gene polymorphic sites including an rs8099917 site and an rs12979860 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site.

2. The hepatitis C virus detection kit according to claim 1, wherein the amplification primer sequence of the rs8099917 site is as shown in SEQ ID NO: 1 to 2.

3. The hepatitis C virus detection kit of claim 1, wherein the amplification primer sequence of the rs12979860 locus is as shown in SEQ ID NO: 3 to 4.

4. The hepatitis C virus detection kit according to claim 1, wherein the multiplex PCR mixed reaction solution includes DNA polymerase and Mg²⁺And dNTPs.

5. The hepatitis C virus detection kit according to claim 4, wherein the multiplex PCR mixed reaction solution further comprises a PCR stabilizer and an enhancer.

6. The hepatitis C virus detection kit of claim 1, wherein the detection kit further comprises a whole blood sample PCR buffer solution, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl, and has a pH of 9.3-9.5.

7. The hepatitis C virus detection kit according to claim 1, wherein the kit amplification system is a 50 μ L PCR system, and the system contains amplification primers of the rs8099917 site and the rs12979860 site.

8. The hepatitis C virus detection kit according to claim 1, wherein the kit amplification system specifically comprises:

2×Platinum Multiplex PCR Master Mix 25μL；

2 mu L of template;

9. The hepatitis C virus detection kit according to claim 1, wherein the kit reaction conditions are pre-denaturation at 95 ℃ for 10 min; then denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 90s, and extension at 72 ℃ for 60s, after 40 cycles, extension at 72 ℃ is carried out again for 10min, and the temperature is restored to 4 ℃.

10. The hepatitis C virus detection kit of claim 1, wherein the detection kit is for use in detecting the onset and/or prognosis of hepatitis C.