CN110438262A - A kind of hepatitis type B virus parting and drug resistant gene detection kit - Google Patents

A kind of hepatitis type B virus parting and drug resistant gene detection kit Download PDF

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CN110438262A
CN110438262A CN201910692625.1A CN201910692625A CN110438262A CN 110438262 A CN110438262 A CN 110438262A CN 201910692625 A CN201910692625 A CN 201910692625A CN 110438262 A CN110438262 A CN 110438262A
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primer
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CN110438262B (en
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蒋析文
黄桃生
李欣钰
郑若楠
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Daan Gene Co Ltd Zhongshan University
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Abstract

The present invention provides a kind of hepatitis type B virus parting and drug resistant gene detection kits, specifically multiple asymmetric PCR technology and electrochemical gene sensor method detection hepatitis type B virus type and the relevant detection kit in drug resistance site and its detection method are utilized the invention discloses a kind of, the experimental results showed that, detection method and kit detection accuracy of the invention be good, specific high, easy to operate automation, the higher advantage of flux, can provide important references for hepatitis B patient medication.

Description

A kind of hepatitis type B virus parting and drug resistant gene detection kit
Technical field
The invention belongs to field of biotechnology, specifically, the present invention relates to a kind of hepatitis type B virus parting and drug resistances Gene detecting kit.
Background technique
Hepatitis type B virus (HBV) is a kind of DNA virus, can cause human liver lesion, be cause cirrhosis, liver cancer and The main reason for chronic hepatitis occurs.It is a serious global hygienic issues.Estimate according to WHO, the whole world there are 2.5 hundred million B-type hepatitis Malicious the infected's (hepatitis b surface antigen positive), hepatitis B in 2015 cause 88.7 ten thousand people dead.
China is hepatitis B big country, and patient accounts for a quarter of global patient.HBV has had found 9 genotype (A~I), Distribution has region.It is dominant with B, C genotype in China Middle Eastern；Southern most area epidemic strain be B2, C2, C1 hypotype, and have small part D genotype；West area especially Xinjiang region is based on D genotype.The study found that HBV hypotype Classification and the selection of the pathogenic of HBV, clinical manifestation and antiviral treatment have certain correlation.Therefore, to HBV The serum or blood plasma of the infected first carries out phenotypic analysis, can play important directive function for clinical treatment.And at the same time, one A more acute problem is just jeopardizing the vast hepatitis B patient of China, and the patient that China has about 35% or more generates second The drug resistance of liver nucleoside medicine, hepatitis B drug resistance have become the current maximum challenge in treating hepatitis B field.
Currently, clinical use treatment hepatitis type B virus nucleosides (acid) analog drug be mainly Lamivudine (LAM), Adefovirdipivoxil (ADV), Entecavir (ETV) and Sebivo (LdT) reduce disease by the duplication of these Drug inhibitions virus Malicious carrying capacity.These drugs all treating hepatitis B poison, patient can not need Long-term taking medicine to carry out maintenance therapy completely.Since patient is long-term Using Anti-HBV activity therapeutic agent, different degrees of medicament-resistant mutation can occur for HBV virus.In treating hepatitis B, drug resistance situation is once sent out Raw, the suppressing virus replication ability of effective antiviral drugs just will be greatly reduced originally.Meanwhile to will lead to the state of an illness anti-for drug resistance The adverse consequences such as multiple, progression of disease, and the crossing drug resistant between drug also can bring great difficulty to the selection of successive treatment. The drug resistant inspection of hepatitis B mainly checks whether the hepatitis B of body generates variation and drug resistance, resistance to by hepatitis B The inspection of medicine variation, we are recognized that the quantity of hepatitis B, replicate situation, communicable strong and weak and be suitble to which is taken A kind of drug is more effective etc..To lay a solid foundation for the therapy rehabilitation of hepatitis B.Therefore B-type hepatitis is clinically detected Malicious resistant mutational site is important to adjusting therapeutic scheme in time, clinical application and real time monitoring antiviral drugs curative effect being instructed to have Directive significance.
Currently used parting and SNP mutation loci detection method have real-time fluorescence PCR method, PCR-Sanger PCR sequencing PCR, Chip method, PCR- high-resolution solubility curve (HRM) method etc..
Real-time fluorescence PCR method high sensitivity, parting is accurate, simple and efficient to handle, and instrument is easy to popularize, easy to spread It uses.But this method flux is not high, and the testing cost of probe higher cost, single locus is related with sample size, and sample size is smaller, Cost is higher.
PCR-Sanger PCR sequencing PCR belongs to qualitative detection, and advantage is that sequencing length is longer, it is possible to find new variant sites.It is main Want insufficient: sensitivity is not high, especially when carrying out tumor tissues somatic mutation detection, when target gene is mutated ratio in tissue When example is lower than 20%, in fact it could happen that false negative result；There is particular/special requirement to reagent and instrument, is not easy to popularize；It is complicated for operation, cost Relatively high, speed is slow, flux is low.
Chip method is mainly reverse dot blot hybridization technique, and the instrument that this method uses is simple, but hybrid process takes a long time, Complicated for operation, sensitivity is low；
PCR- high-resolution melting curve (HRM) method is easy to operate, quick, flux is big, use cost is low, result is accurate, It is advantageously implemented stopped pipe operation, can determine the height of methylation according to melting curve when carrying out DNA methylation assay.The party The shortcomings that method, is: can not rule out emerging hereditary variation in determined nucleic acid；Since single base mutation leads to DNA melting temperature Variation it is very small, this method has higher requirements to the sensitivity of instrument and resolution ratio.
Therefore, it is high, easy to operate to be dedicated to developing accuracy by those skilled in the art, can detect several genes type simultaneously With the new molecular diagnosis method in drug resistance site, to instruct clinical application.
Summary of the invention
The purpose of the present invention is to provide a kind of hepatitis type B virus parting and drug resistant gene detection kits.
The first aspect of the present invention provides what one kind was detected for hepatitis type B virus (HBV) parting and drug resistant gene For PCR primer to group, the primer pair group includes the first primer pair, and the first primer is to including as shown in SEQ ID NO.:1 Forward primer；With the reverse primer as shown in SEQ ID NO.:2.
In another preferred example, the primer pair group further includes the second primer pair, and second primer pair includes such as SEQ Forward primer shown in ID NO.:3；With the reverse primer as shown in SEQ ID NO.:4.
In another preferred example, the primer pair group further includes third primer pair, and the third primer pair includes such as SEQ Forward primer shown in ID NO.:5；With the reverse primer as shown in SEQ ID NO.:6.
In another preferred example, the primer pair group further includes the 4th primer pair, and the 4th primer pair includes such as SEQ Forward primer shown in ID NO.:7；With the reverse primer as shown in SEQ ID NO.:8.
In another preferred example, the primer pair group further includes the 5th primer pair, and the 5th primer pair includes such as SEQ Forward primer shown in ID NO.:9；With the reverse primer as shown in SEQ ID NO.:10.
In another preferred example, the primer pair group further includes the 6th primer pair, and the 6th primer pair includes such as SEQ Forward primer shown in ID NO.:54；With the reverse primer as shown in SEQ ID NO.:55.
The second aspect of the present invention provides what one kind was detected for hepatitis type B virus (HBV) parting and drug resistant gene Signal probe group, the signal probe group include one or more signal probes selected from the group below:
173WSP:TATGGGAGTGGGCCTCA, SEQ ID NO.:11；
173MSP:TATGGGATTGGGCCT, SEQ ID NO.:12；
180MSP:GTTTCTCATGGCTCA, SEQ ID NO.:13；
180WSP1:CGTTTCTCTTGGCTCAG, SEQ ID NO.:14；
180WSP2:GTTTCTCCTGGCTCAGT, SEQ ID NO.:15；
181MSP1:TTCTCATGGTTCAGTTTAC, SEQ ID NO.:16；
181MSP2:TTTCTCCTGACTCAGTTTA, SEQ ID NO.:17；
194WSP1:CGTAGGGCATTCCC, SEQ ID NO.:56；
194WSP2:CGCCGGGCTTTC, SEQ ID NO.:57；
194MSP:CGCCGGACTTTCC, SEQ ID NO.:58；
204WSP:TTCAGTTATATGGATGATG, SEQ ID NO.:18；
204MSP1:TCAGTTATGTGGATGAT, SEQ ID NO.:19；
204MSP2:CAGTTATATAGATGATGTG, SEQ ID NO.:20；
204MSP3:CAGTTATATTGATGATGTG, SEQ ID NO.:21；
204MSP4:CAGTTATATCGATGATGTG, SEQ ID NO.:22；
236WSP1:ACATTTGAACCCTAATA, SEQ ID NO.:23；
236WSP2:ACATTTGAATCCTCATA, SEQ ID NO.:24；
236MSP:ACATTTAACCCCTCACA, SEQ ID NO.:25；
250WSP:AAATTTCATGGGTTATGT, SEQ ID NO.:26；
250MSP:TTAATTTCGTGGGATAT, SEQ ID NO.:27；
B-SP:TCCCAAATCTCCAGTCA, SEQ ID NO.:28；
C-SP:GAGCACCCACGT, SEQ ID NO.:29；With,
D-SP:ATCTTTCCACCAGCAAT, SEQ ID NO.:30.
In another preferred example, the signal probe group further includes signal probe:
NB-SP:GCATCTTCAAACTCAAA, SEQ ID NO.:31.
The third aspect of the present invention provides what one kind was detected for hepatitis type B virus (HBV) parting and drug resistant gene Capture probe group, the capture probe group include one or more capture probes selected from the group below:
173CP:TGGGCTTTCGCAAGATTCCTAT, SEQ ID NO.:32
180CP:ATGGGAGTGGGCCTCAGT, SEQ ID NO.:33
194CP:AGTGCCATTTGTTCAGTGGTT, SEQ ID NO.:59
204CP:TTCCCCCACTGTCTGGCTTT, SEQ ID NO.:34
236CP:TTTCTTTTGTCTTTGGGTAT, SEQ ID NO.:35
250CP:ACGTTGGGGCTACTCCCT, SEQ ID NO.:36
B-Cp:TGTCTTGGCCAAAATTCGCAG, SEQ ID NO.:37
C-Cp:TGGACTTCTCTCAATTTTCTAGG, SEQ ID NO.:38, and
D-Cp:AGCTACAGCATGGGGCAGA, SEQ ID NO.:39.
In another preferred example, the capture probe group further includes capture probe:
NB-CP:ATCTATTGCTTACATTTGCTT, SEQ ID NO.:40.
The fourth aspect of the present invention provides what one kind was detected for hepatitis type B virus (HBV) parting and drug resistant gene Kit, the kit include PCR primer described in first aspect present invention to group.
In another preferred example, the kit further includes signal probe group described in second aspect of the present invention.
In another preferred example, the kit further includes capture probe group described in third aspect present invention.
In another preferred example, the kit further includes one or more components selected from the group below:
Hot start Taq polymerase, UDG enzyme, dNTPs, Tris-HCl, MgCl2、(NH4)2SO4And Tween-20.
In another preferred example, the kit further includes negative quality-control product.
In another preferred example, the kit further includes internal standard quality-control product.
The fourth aspect of the present invention provides the method for hepatitis type B virus (HBV) parting and drug resistant gene detection, The method includes the steps:
(1) sample to be detected is provided, and extracts viral nucleic acid from the sample to be detected；
(2) step (1) is extracted to obtained viral nucleic acid, the multiple asymmetric PCR amplification of PCR reaction solution progress is added, respectively Obtain pcr amplification product；
It wherein, include primer pair group described in first aspect present invention in the PCR reaction solution；
(3) PCR product hybridization check
It is added in Electrochemistry gene chip after pcr amplification product is mixed with electrochemical hybridization liquid, in electrochemical gene It is detected in chip.
In another preferred example, in the step (2), the first primer centering, forward primer shown in SEQ ID NO.:1 Application percentage with reverse primer shown in SEQ ID NO.:2 is 0.15:0.75.
In another preferred example, in the step (2), in the second primer pair, forward primer shown in SEQ ID NO.:3 Application percentage with reverse primer shown in SEQ ID NO.:4 is 0.25:1.25.
In another preferred example, in the step (2), in third primer pair, forward primer shown in SEQ ID NO.:5 Application percentage with reverse primer shown in SEQ ID NO.:6 is 0.15:1.0.
In another preferred example, in the step (2), in the 4th primer pair, forward primer shown in SEQ ID NO.:7 Application percentage with reverse primer shown in SEQ ID NO.:8 is 0.1:0.5.
In another preferred example, in the step (2), in the 5th primer pair, forward primer shown in SEQ ID NO.:9 Application percentage with reverse primer shown in SEQ ID NO.:10 is 0.1:0.5.
In another preferred example, in the step (2), in the 6th primer pair, forward primer shown in SEQ ID NO.:61 Application percentage with reverse primer shown in SEQ ID NO.:62 is 0.1:0.5.
In another preferred example, the PCR amplification condition are as follows: 50 DEG C 3 minutes, 95 DEG C initial denaturation 15 minutes, then press 94 45 circulations of DEG C of 30 seconds → 55 DEG C 30 seconds → 72 DEG C amplifications in 30 seconds, last 72 DEG C extend 7 minutes.
In another preferred example, the electrochemical hybridization liquid includes signal probe group described in second aspect of the present invention.
In another preferred example, the electrochemical hybridization liquid includes: electrochemical hybridization liquid I, NBS buffer and NaClO4, Wherein, electrochemical hybridization liquid I includes signal probe group and MES buffer described in second aspect of the present invention.
In another preferred example, the concentration of each signal probe is 0.2 μM in the electrochemical hybridization liquid I.
In another preferred example, the Electrochemistry gene chip includes capture probe group described in third aspect present invention.
In another preferred example, the method is non-diagnostic purpose.
The fifth aspect of the present invention provides primer pair group and second aspect of the present invention described in first aspect present invention The purposes of the probe groups, is used to prepare detection kit, and the detection kit is used for hepatitis type B virus (HBV) parting With the detection of drug resistant gene detection.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and in below (eg embodiment) specifically It can be combined with each other between each technical characteristic of description, to form a new or preferred technical solution.As space is limited, herein No longer tire out one by one and states.
Detailed description of the invention
Fig. 1 shows negative sample testing result.
Fig. 2 shows Type B pattern detection result.
Fig. 3 shows c-type pattern detection result.
Fig. 4 shows this testing result of D pattern.
Fig. 5 shows this testing result of B/C pattern.
Fig. 6 shows rtL180M and rtM204V sudden change sample testing result.
Fig. 7 shows rtA181T/V sudden change sample testing result.
Fig. 8 shows that 1 ratio of primer pair adjusts each site signal value situation of change.
Fig. 9 shows different c-type primer pairs each site signal value situation of change in multiple system.
Figure 10 shows different primers to 1 each site signal value situation of change of amplification.
Specific embodiment
The present inventor is obtained a kind of using multiple asymmetric PCR technology and electrochemical-based by extensive and in-depth research Because sensor method detects hepatitis type B virus type and the relevant detection kit in drug resistance site and its detection method, experimental result Show that good detection method and kit detection accuracy of the invention, specific high, easy to operate automation, flux are higher Advantage can provide important references for hepatitis B patient medication.Kit and detection method of the invention, can be widely applied The window phase detection of hepatitis B caused by HBV virus, clinical diagnosis, scientific research, curative effect such as track at the multiple fields.
Before describing the present invention, it should be understood that the present invention is not limited to the specific method and experiment conditions, because this Class method and condition can change.It should also be understood that its purpose of the term as used herein is only that description specific embodiment, and And it is not intended to be restrictive, the scope of the present invention will be limited only by the claims which follow.
Unless otherwise defined, otherwise whole technologies used herein and scientific term all have such as fields of the present invention The normally understood identical meanings of those of ordinary skill.As used herein, in use, term in mentioning the numerical value specifically enumerated " about " mean that the value can change not more than 1% from the value enumerated.For example, as used herein, statement " about 100 " includes 99 Hes 101 and between whole values (for example, 99.1,99.2,99.3,99.4 etc.).
Although can be used in implementation or test of the invention and heretofore described similar or of equal value any method And material, place enumerates preferred method and material herein.
Electrochemistry gene chip method
Electrochemistry gene chip method (CN201310166271.X, CN201210590669.1) passes through electrochemical gene core Piece analysis system detects current value (signal value), does not determine the signal value of each point, can be easily accomplished middle flux Detection.In addition, its is cheap, equipment is simply light and small, easy to operate, tests quick and precisely.In the present invention, a kind of base is established The other and related medicament-resistant mutation base of genotype of hepatitis B virus is detected simultaneously in multiple asymmetric PCR-Electrochemistry gene chip method Recruit's diagnostic method of cause, and develop its kit, parting to hepatitis type B virus and instructs clinical application to have weight Want meaning.
Electrochemical gene sensor chip is using special chemical method treated printed circuit board as carrier, by various probes Or target fragments are fixed on printed circuit board surface in a manner of chemical bonds, and using ferrocene derivatives as electrochemistry Indicator forms the micro-array chip that can be used for hybridization reaction or antigen-antibody reaction.
Multiple asymmetric PCR
Multiplex PCR (multiplex PCR), also known as Multiplex PCR or composite PCR, it is in same PCR reaction system In add two pairs or more primers, while amplifying the PCR reaction of multiple nucleic acid fragments, reaction principle, reaction reagent and operation Process is identical as general PCR.
Asymmetric PCR (asymmetric PCR) is the pair of primers with inequality, is generated after PCR amplification a large amount of single-stranded DNA(ssDNA).This is referred to as unrestricted primer and restricted primer to primer, and ratio is preferably 5-20: 1.It is anti-in PCR In the initial 10-15 circulation answered, amplified production is mainly double-stranded DNA, but when restricted primer (low concentration primer) consumes After complete, the PCR of non-limiting primer (high density primer) guidance will generate a large amount of single stranded DNA.The key of asymmetric PCR is The absolute magnitude of restricted primer is controlled, the ratio of two primers need to be repeatedly optimized.Still an alternative is that first drawing with isoconcentration Object PCR amplification, prepares double-stranded DNA, (dsDNA), then using this dsDNA as template, then with a primer therein progress second Secondary PCR prepares ssDNA.The ssDNA of asymmetric PCR preparation, is mainly used for determining nucleic acid sequence.
The many because being known as of multiple asymmetric PCR reaction are influenced, such as:
(1) reaction system is uneven, and the imbalance of reaction system leads to certain advantage primers in several wheels reaction of early period And its template expands rapidly, obtains a large amount of amplified production, and these amplified productions are the good inhibition of archaeal dna polymerase simultaneously Agent.So the polymerizing power of polymerase is by more more and more intense inhibition, therefore, at early period with a large amount of appearance of amplified production In the primer and its template of disadvantage, at this moment just more it is difficult to react, it is very small to eventually lead to amplified production amount, so that it cannot Detection.
(2) primer specificity, if primer and the non-target gene fragment binding force of other in system are stronger, purpose base It is striven unexpectedly because combining the ability of primer just to will receive, so as to cause amplification efficiency decline.
(3) optimum annealing temperature is inconsistent, and multipair primer is placed into a system and is expanded, due to carrying out PCR reaction Annealing temperature it is identical, it requires that the optimum annealing temperature of every pair of primers is close.
(4) primer dimer, including between primer dimer and primer itself be formed by hairpin structure, there are also a kind of It is the aggressiveness that third party DNA is mediated, these dimers and non-specific primer is the same can all interfere primer and target binding site Unexpectedly it strives, influences amplification efficiency.
Although above-mentioned be referred to the several factors for influencing amplification efficiency, more factors are unclear.Up to the present, There are no the effective ways that one can clearly predict amplification efficiency.
The present inventor, not with related drug resistance site, carries out after going deep into comparing analysis, designs to genotype of hepatitis B virus Primer and probe, then the primer and probe of design is in optimized selection and verified, finally determined can be used for it is multiple not The primer and probe sequence of non-symmetric PCR amplification, provide hepatitis type B virus parting and drug resistant mutant genes on this basis PCR- Electrochemistry gene chip method detection kit.
The present inventor in the course of the research, not and related drug resistance site PCR amplification primer to genotype of hepatitis B virus Design and experimental verification are carried out.The result shows that same can make to detect HBV gene type and related drug resistance position using a tube body system Point.DNA biochip of electrochemical gene sensor is the energy letter for being combined into nucleic acid hybridization technique and electrochemical sensor technology The new method of single quick, accurate inexpensively assisted diagnosis patient disease.The technology will on support (electrode) regular row Column secure a large amount of ssDNA probe, constitute two-dimensional ssDNA probe array, use is engaged with electrochemical techniques, can be right simultaneously A large amount of DNA is tested and analyzed, and easy to operate, detection efficiency is high and high degree of automation.
The present invention provides a kind of easy to operate, detection efficiency it is high be used for hepatitis type B virus type and medicament-resistant mutation base Because of detection kit, can disposably detect tri- gene types of B, C, D and rt173, rt180, rt181, rt194, rt204, 6 drug resistance sites in rt236, rt250.Wherein, rt173 drug resistance site mutations are that rtV173L, rt180 drug resistance sites are prominent It is rtA194T, rt204 that become rtL180M, rt181 drug resistance site mutations, which be rtA181T/V, rt194 drug resistance site mutations, It is that rtN236T, rt250 drug resistance site mutations are that position drug resistance site mutation, which is rtM204V/I, rt236 drug resistance site mutations, rtM250V.It can refer to document about each medicament-resistant mutation: Leandro R, et al.Hepatitis B virus resistance substitutions:long-term analysis by next-generation sequencing[J].Arch Virol, 2016,161 (10): 2885-2891. opens vivid etc., 147 chronic hepatitis Bs HBV P after nucleosides or thuja acid class drug therapy Gene regions mutation analysis [J] .2015,12,019.
It is preferably carried out in mode, provides a kind of based on multiple asymmetric PCR-electrochemical gene at of the invention one Sensor method detects hepatitis type B virus type and drug resistant mutant genes detection kit, including reaction solution A, enzyme system, hybridization solution I、NBS、NaClO4；Specific each PCR primer is as shown in table 1:
1 primer mark sheet of table
Above-mentioned each PCR primer efficiently solves that high GC content sequence PCR amplification is invalid, inefficient or non-specific amplification Problem, and also have good applicability for non-high GC content sequence PCR amplification.
Oligonucleotides signal probe in above-mentioned hybridization solution I comprising one group with PCR product specific binding, sequence such as table 2:
2 signal probe mark sheet of table
Remarks: " W " represents wild type, and " M " represents saltant type；
It is of the invention based on multiple asymmetric PCR-electrochemical gene sensor method detection hepatitis type B virus parting and resistance to The kit of medicine genetic test, special printed circuit board gold electrode table can be fixed in the form of covalent bond by further including one group The oligonucleotide capture probe in face, sequence such as table 3:
3 capture probe mark sheet of table
CP probe title CP probe sequence (5 ' -3 ') SEQ ID NO.:
173CP TGGGCTTTCGCAAGATTCCTAT 32
180CP ATGGGAGTGGGCCTCAGT 33
194CP AGTGCCATTTGTTCAGTGGTT 59
204CP TTCCCCCACTGTCTGGCTTT 34
236CP TTTCTTTTGTCTTTGGGTAT 35
250CP ACGTTGGGGCTACTCCCT 36
B-Cp TGTCTTGGCCAAAATTCGCAG 37
C-Cp TGGACTTCTCTCAATTTTCTAGG 38
D-Cp AGCTACAGCATGGGGCAGA 39
NB-CP ATCTATTGCTTACATTTGCTT 40
3 ' ends of above-mentioned capture probe are marked with C6 S-S, and special printed circuit board is fixed in the form of covalent bond Gold electrode surfaces, for capturing PCR product；5 ' ends of above-mentioned signal probe are marked by different ferrocene markers, With captured PCR product specific hybridization, apply alternating voltage on the electrode, redox reaction then occurs for ferrocene, passes through Current value is detected to determine result yin and yang attribute.The Crossing system of this PCR product and double probes, ensure that this Electrochemistry gene chip The good specificity of method.
It is preferably carried out in mode at of the invention one, above-mentioned hepatitis type B virus parting and drug resistance according to the present invention The specific ingredient of gene detecting kit and dosage such as the following table 4:
4 PCR reaction solution formula of table
The present invention also provides PCR- electrochemical gene sensor method detection hepatitis type B virus parting and medicament-resistant mutation bases Because of the method for detection, specifically includes the following steps:
(1) extracts kit progress genomic nucleic acids extraction is recommended in sample collection and use；
(2) it takes 20 μ l that PCR reaction solution is added sample genomic dna after extraction and carries out multiple asymmetric PCR amplification, PCR Reaction condition are as follows: 50 DEG C 3 minutes, 95 DEG C initial denaturation 15 minutes, then by 94 DEG C 30 seconds → 55 DEG C 30 seconds → 72 DEG C 30 seconds expand 45 circulations, last 72 DEG C extend 7 minutes；
(3) 70 μ l hybridization solution I, 10 μ l NBS, 20 μ l NaClO are added into pcr amplification product4It mixes well, is transferred to In the pipe of electrochemical sensor, compressing pipe lid.Then it inserts the sensors into electrochemical apparatus to be detected, obtains result.
Main advantages of the present invention are:
It is provided by the invention a kind of based on multiple asymmetric PCR-electrochemical gene sensor method detection hepatitis type B virus The kit of parting and drug resistant mutant genes detection, good, specific high, easy to operate automation, flux with detection accuracy Higher advantage provides important references for hepatitis B patient medication guide.
Combined with specific embodiments below, the further old present invention in detail.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.The experimental method of detailed conditions is not specified in the following example, usually according to conventional strip Part such as U.S. Sambrook.J etc. writes " Molecular Cloning: A Laboratory room guide " (Huang Peitang etc. is translated, Beijing: Science Press, 2002) Described in condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number be by weight It calculates.Experimental material used in following embodiment and reagent can obtain unless otherwise instructed from commercially available channel.
The present invention establishes by multiple asymmetric PCR-electrochemical gene sensor method and is directed to hepatitis type B virus B, C, D The quick side of detection of genotype and the site rt173, rt180, rt181, rt194, rt204, rt236, rt250 drug resistant mutant genes Method.
The optimization and determination of embodiment 1 hepatitis type B virus parting and drug resistant mutant genes detection kit
The design and optimization of 1.1 primers:
The sequence inquired and downloaded using ncbi database, design primer have simultaneously carried out a large amount of screening study work, finally Each PCR primer and multi-PRC reaction system in above-mentioned table 1 has been determined.
The optimization of primer concentration: orthonormal design of experiments is utilized, upstream and downstream primer amount ratio is by 1/5 in 50 μ l reaction systems Agarose gel electrophoresis and PCR reaction test are carried out to 5/50, is tested through being repeated several times, finally determines the work of optimal each primer It is as shown in Table 4 above to make concentration.
1.2 reaction system optimizations:
The optimization of hot start Taq polymerase dosage: orthonormal design of experiments is utilized, is used respectively in 50 μ l reaction systems from 5U Enzyme dosage/reaction of (enzyme unit) to 15U concentration gradient carries out PCR reaction, tests, finally determines optimal through being repeated several times Taq enzyme dosage is 10U/ reaction.
The optimization of UDG enzyme dosage: orthonormal design of experiments is utilized, is used respectively in 50 μ l reaction systems from 0.05U (enzyme list Position) to enzyme dosage/reaction progress PCR reaction of 1U concentration gradient, it is tested through being repeated several times, finally determines that optimal UDG enzyme is used Amount is that 0.75U/ reacts.
The optimization of dNTPs concentration: orthonormal design of experiments is utilized, is used respectively in 50 μ l reaction systems from 0.1mmol/L DNTPs to 1mmol/L concentration gradient carries out PCR reaction, tests through being repeated several times, finally determines that optimal dNTPs concentration is 0.334mmol/L。
The optimization of template sample-adding amount: it in the case that other components are constant in the reaction system, is tested respectively from 10 μ l to 20 μ The sample-adding amount of l gradient carries out PCR reaction, tests through being repeated several times, and finally determines that best sample-adding amount is 20 μ l.
The optimization of reaction temperature: according to the length of the activity of enzyme and few polynucleotides, mainly to annealing temperature and when extending Between be optimized, tested through being repeated several times, finally determine optimal reaction temperature and time are as follows: 50 DEG C 3 minutes, 95 DEG C pre- to become Property 15 minutes；Then 94 DEG C 30 seconds, 55 DEG C 30 seconds, 72 DEG C 30 seconds, 45 circulation；Last 72 DEG C extend 7 minutes.
1.3 kits detection limit determines:
Selection has been demarcated as 1*106IU/mL hepatitis type B virus B, C, D type nucleic acid gradient dilution is to 1*105IU/mL、1* 104IU/mL、1*103IU/mL、1*102IU/mL、1*101IU/mL, each concentration are repeated 10 times respectively, carry out multiple asymmetric PCR After qualitative amplification, hybridization check is carried out in electrochemical gene sensor detection system, testing result is shown, when viral nucleic acid is dense Degree is 1*101When IU/mL, moiety site no signal；And when DNA concentration is 1*102IU/mL to 1*105When IU/mL, through Sanger The testing result that each site is checked in sequencing is correct.Therefore, this kit detection limit is determined as 1*102IU/mL。
2 clinical sample of embodiment detects materials and methods
2.1 viral DNA nucleic acid extractions
Sample requirement:
(1) specimen types are applicable in: being clearly serum, the plasma sample of the patient of the hbv nucleic acid positive.
(2) collection of specimens: 1. serum: extracting subject's venous blood 2mL with disposable sterilized injector, injects sterile dry Dry glass tube, (22~25 DEG C) of room temperature are placed 30~60 minutes, blood specimen can spontaneous complete agglutination serum, or directly use is precipitated Horizontal centrifuge, 4000rpm are centrifuged 5 minutes；Upper serum is drawn, 1.5mL sterile centrifugation tube is transferred to；2. blood plasma: with once Property asepsis injector extract subject's venous blood 2mL, injection contain EDTA (disodium ethylene diamine tetraacetate) or sodium citrate anticoagulant Glass tube, gently overturn glass tube immediately and mix 5~10 times, mix well anti-coagulants with venous blood, 5~be after ten minutes It may separate out blood plasma, be transferred to 1.5mL sterile centrifugation tube.
(3) Saving specimen and transport: sample can be immediately available for testing；It is 12 months in -20 ± 5 DEG C of storage lives；To length Phase saves, and need to be stored in -80 ± 5 DEG C；Multigelation should be avoided in sample.Sample transport is on the rocks using curling stone or bubble chamber is on the rocks close Envelope transport, haulage time are not to be exceeded 4 days.
Operating procedure:
60 hepatitis B patient plasma samples are acquired, the core using Da'an Gene Company, Zhongshan University's production is recommended Sour extracts kit (such as: nucleic acid extraction purification kit (centrifugal column method), nucleic acid extraction purification kit (paramagnetic particle method)) carries out Genomic DNA is extracted, concrete operation step is please guided according to kit specification and carried out.
2.2 multiple asymmetric PCR amplifications and electrochemical hybridization detection
Sample genomic dna after extraction is separately added into 25 μ l and carries out multiple asymmetric PCR into reaction solution described above Amplification, PCR reaction condition are as follows: 50 DEG C 3 minutes, 95 DEG C initial denaturation 15 minutes, then press 94 DEG C 30 seconds → 55 DEG C 30 seconds → 72 DEG C 45 circulations of amplification in 30 seconds, last 72 DEG C extend 7 minutes；Sequentially added in pcr amplification product 70 μ l signal probe mixed liquors, 10μl NBS、20μl NaClO4It mixes well, is transferred in electrochemical sensor, compressing pipe lid.Then electricity is inserted the sensors into Chemical apparatuses is detected, and result is obtained.
The analysis of 2.3 electrochemical results
It is B-mode to 50 using hepatitis type B virus parting established by the present invention and drug resistant mutant genes detection kit Hepatitis virus patient clinical sample is detected, and using the PCR sequencing PCR inspection of Da'an Gene Company, Zhongshan University's production Test agent box carries out control verifying, as the result is shown in 50 hepatitis B patient samples, positive rate 100%, and two kinds of sides Method consistency is high, as a result has statistical significance.
Specific to different types and drug resistance site, Type B 17, c-type 29,4, D type, wherein 1, the site rt173, 5, the site rt180,2, the site rt181,8, the site rt204,1, the site rt236,1, the site rt250, are not detected Rt194 site mutation does not occur missing inspection situation.
Fig. 1 shows negative sample testing result.2 show Type B pattern detection result.Fig. 3 shows c-type pattern detection As a result.Fig. 4 shows this testing result of D pattern.Fig. 5 shows this testing result of B/C pattern.Fig. 6 show rtL180M and RtM204V sudden change sample testing result.Fig. 7 shows rtA181T/V sudden change sample testing result.
Comparative example 1
The present inventor to tri- gene types of hepatitis type B virus B, C, D and rt173, rt180, rt181, rt194, 7 drug resistance sites in rt204, rt236, rt250 carry out after going deep into comparing analysis, and it is tens of right to devise for each target sequence Primer and tens of kinds of probes, due to reaction system imbalance, primer specific sex differernce, annealing temperature be inconsistent and primer two The reasons such as aggressiveness are difficult to obtain effective with multiple asymmetric PCR amplimer and probe sequence.
The present inventor is in optimized selection and verifies through a large number of experiments, to the primer and probe of design, final to determine It can be used for primer, probe sequence and combinations thereof of multiple asymmetric PCR amplification.
Even if being added not year-on-year in the case where determining the primer pair and probe sequence that are directed to each target nucleic acid substantially The primer row multiplex amplification of example, there is also differences for effect, it is therefore desirable to optimize to forward and reverse primer ratio.
Fig. 8 shows that 1 ratio of primer pair adjusts each site signal value situation of change.
For example, the ratio of the forward and reverse primer of primer pair 1 is adjusted to 0.15:1 in multiple asymmetric PCR step, detect As a result the signal value for the site of detection site 236 and 250 of primer pair 2 has apparent decreasing trend.
The optimization of 2 c-type primed probe of comparative example
The present inventor devises tens of pairs of primers and tens of kinds of probes for each target sequence, this comparative example by taking c-type as an example, Illustrate the undesirable primer and probe of part effect.
Compare primer pair 1:
2F-1:CAAGAGCTACAGCATGGGA, SEQ ID NO.:41；
2R-1:GTGATCCTTGTTGGGGTT, SEQ ID NO.:42；
Compare primer pair 2:
2F-2:CAAGAGCTACAGCATGGGA, SEQ ID NO.:41；
2R-2:CTGTTGTCAAAATGCCCTG, SEQ ID NO.:43；
Compare primer pair 3:
2F-3:TCGCAGAAGATCTAAATCTCGG, SEQ ID NO.:44；
2R-3:ACAAACCAGATTGGGACT, SEQ ID NO.:45；
Compare primer pair 4:
2F-4:TCGCAGAAGATCTAAATCTCGG, SEQ ID NO.:44；
2R-4:TCAGGGCATACTACAAAC, SEQ ID NO.:46；
Compare primer pair 5:
2F-5:TCGCAGAAGATCTAAATCTCGG, SEQ ID NO.:44；
2R-5:CTGTTGTCAATATGCCCTG, SEQ ID NO.:47；
Fig. 9 shows different c-type primer pairs each site signal value situation of change in multiple system.Above-mentioned several pairs of primers Signal value is lower, and is added in multiple system and has inhibiting effect to the signal value of other primers, is unable to satisfy clinic and answers It is required that
The primer of 3 primer pair 1 of comparative example optimizes
The present inventor devises tens of pairs of primers and tens of kinds of probes for each target sequence, this comparative example is with primer pair 1 For, illustrate the undesirable primer of part effect.
Compare primer pair 1 ':
3F-1:ACTTGTATTCCCATCCCAT, SEQ ID NO.:48；
3R-1:GACTCAAGATGTTGTACAGACTTGG, SEQ ID NO.:49；
Compare primer pair 2 ':
3F-2:ACCTCTATGTTTCCCTCATGTTGCT, SEQ ID NO.:50；
3R-2:GACTCAAGATGTTGTACAGACTTGG, SEQ ID NO.:49；
Compare primer pair 3 ':
3F-3:ACTTGTATTCCCATCCCAT, SEQ ID NO.:48；
3R-3:CGGCATAAAGGGACTCAAGATGT, SEQ ID NO.:51；
Compare primer pair 4 ':
3F-4:ACCTCTATGTTTCCCTCATGTTGCT, SEQ ID NO.:50；
3R-4:CGGCATAAAGGGACTCAAGATGT, SEQ ID NO.:51；
Compare primer pair 5 ':
3F-5:TTCGCAARATTCCTATGGGAGT, SEQ ID NO.:52；
3R-5:GAACCACTGAACAAATGGCAC, SEQ ID NO.:53；
Figure 10 shows different primers to 1 each site signal value situation of change of amplification.The signal value of the above primer pair exists Between 10-20, PCR amplification effect is poor, is unable to satisfy the requirement of clinical application.
All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.
Sequence table
<110>Da'an Gene Company, Zhongshan University
<120>a kind of hepatitis type B virus parting and drug resistant gene detection kit
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<210> 20
<211> 19
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<400> 20
cagttatata gatgatgtg 19
<210> 21
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<400> 21
cagttatatt gatgatgtg 19
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<211> 17
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<400> 25
acatttaacc cctcaca 17
<210> 26
<211> 18
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<400> 26
aaatttcatg ggttatgt 18
<210> 27
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<400> 27
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<400> 28
tcccaaatct ccagtca 17
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<400> 30
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<210> 31
<211> 17
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<400> 31
gcatcttcaa actcaaa 17
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tgggctttcg caagattcct at 22
<210> 33
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atgggagtgg gcctcagt 18
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<400> 34
ttcccccact gtctggcttt 20
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<400> 35
tttcttttgt ctttgggtat 20
<210> 36
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<400> 36
acgttggggc tactccct 18
<210> 37
<211> 21
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tgtcttggcc aaaattcgca g 21
<210> 38
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tggacttctc tcaattttct agg 23
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caagagctac agcatggga 19
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<400> 42
gtgatccttg ttggggtt 18
<210> 43
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<400> 43
ctgttgtcaa aatgccctg 19
<210> 44
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<400> 45
acaaaccaga ttgggact 18
<210> 46
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tcagggcata ctacaaac 18
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<400> 47
ctgttgtcaa tatgccctg 19
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<400> 48
acttgtattc ccatcccat 19
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gactcaagat gttgtacaga cttgg 25
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<400> 50
acctctatgt ttccctcatg ttgct 25
<210> 51
<211> 23
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<400> 51
cggcataaag ggactcaaga tgt 23
<210> 52
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<400> 52
ttcgcaarat tcctatggga gt 22
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gaaccactga acaaatggca c 21
<210> 54
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aggcggggtt tttcttgtt 19
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<400> 55
gcagacacat ccagcgata 19
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<400> 56
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Claims (10)

1. one kind is for hepatitis type B virus (HBV) parting and the PCR primer of drug resistant gene detection to group, which is characterized in that institute Stating primer pair group includes one or more primer pairs selected from the group below:
The first primer pair, the first primer is to including the forward primer as shown in SEQ ID NO.:1；With such as SEQ ID Reverse primer shown in NO.:2；
Second primer pair, second primer pair include the forward primer as shown in SEQ ID NO.:3；With such as SEQ ID Reverse primer shown in NO.:4；
Third primer pair, the third primer pair include the forward primer as shown in SEQ ID NO.:5；With such as SEQ ID Reverse primer shown in NO.:6；With
4th primer pair, the 4th primer pair include the forward primer as shown in SEQ ID NO.:7；With such as SEQ ID Reverse primer shown in NO.:8；
6th primer pair, the 6th primer pair include the forward primer as shown in SEQ ID NO.:54；With such as SEQ ID Reverse primer shown in NO.:55；
Preferably, the primer pair group further includes the 5th primer pair, and the 5th primer pair includes as shown in SEQ ID NO.:9 Forward primer；With the reverse primer as shown in SEQ ID NO.:10.
2. the signal probe group that one kind is detected for hepatitis type B virus (HBV) parting and drug resistant gene, which is characterized in that described Signal probe group includes one or more signal probes selected from the group below:
173WSP:TATGGGAGTGGGCCTCA, SEQ ID NO.:11；
173MSP:TATGGGATTGGGCCT, SEQ ID NO.:12；
180MSP:GTTTCTCATGGCTCA, SEQ ID NO.:13；
180WSP1:CGTTTCTCTTGGCTCAG, SEQ ID NO.:14；
180WSP2:GTTTCTCCTGGCTCAGT, SEQ ID NO.:15；
181MSP1:TTCTCATGGTTCAGTTTAC, SEQ ID NO.:16；
181MSP2:TTTCTCCTGACTCAGTTTA, SEQ ID NO.:17；
194WSP1:CGTAGGGCATTCCC, SEQ ID NO.:56
194WSP2:CGCCGGGCTTTC, SEQ ID NO.:57
194MSP:CGCCGGACTTTCC, SEQ ID NO.:58
204WSP:TTCAGTTATATGGATGATG, SEQ ID NO.:18；
204MSP1:TCAGTTATGTGGATGAT, SEQ ID NO.:19；
204MSP2:CAGTTATATAGATGATGTG, SEQ ID NO.:20；
204MSP3:CAGTTATATTGATGATGTG, SEQ ID NO.:21；
204MSP4:CAGTTATATCGATGATGTG, SEQ ID NO.:22；
236WSP1:ACATTTGAACCCTAATA, SEQ ID NO.:23；
236WSP2:ACATTTGAATCCTCATA, SEQ ID NO.:24；
236MSP:ACATTTAACCCCTCACA, SEQ ID NO.:25；
250WSP:AAATTTCATGGGTTATGT, SEQ ID NO.:26；
250MSP:TTAATTTCGTGGGATAT, SEQ ID NO.:27；
B-SP:TCCCAAATCTCCAGTCA, SEQ ID NO.:28；
C-SP:GAGCACCCACGT, SEQ ID NO.:29；With,
D-SP:ATCTTTCCACCAGCAAT, SEQ ID NO.:30.
3. the capture probe group that one kind is detected for hepatitis type B virus (HBV) parting and drug resistant gene, which is characterized in that described Capture probe group includes one or more capture probes selected from the group below:
173CP:TGGGCTTTCGCAAGATTCCTAT, SEQ ID NO.:32
180CP:ATGGGAGTGGGCCTCAGT, SEQ ID NO.:33
194CP:AGTGCCATTTGTTCAGTGGTT, SEQ ID NO.:59
204CP:TTCCCCCACTGTCTGGCTTT, SEQ ID NO.:34
236CP:TTTCTTTTGTCTTTGGGTAT, SEQ ID NO.:35
250CP:ACGTTGGGGCTACTCCCT, SEQ ID NO.:36
B-Cp:TGTCTTGGCCAAAATTCGCAG, SEQ ID NO.:37
C-Cp:TGGACTTCTCTCAATTTTCTAGG, SEQ ID NO.:38, and
D-Cp:AGCTACAGCATGGGGCAGA, SEQ ID NO.:39.
4. the kit that one kind is detected for hepatitis type B virus (HBV) parting and drug resistant gene, which is characterized in that the reagent Box includes PCR primer described in first aspect present invention to group.
5. kit as claimed in claim 4, which is characterized in that the kit further includes signal as claimed in claim 2 Probe groups；And/or
The kit further includes capture probe group as claimed in claim 3.
6. kit as claimed in claim 4, which is characterized in that the kit further includes selected from the group below one or more Component:
Hot start Taq polymerase, UDG enzyme, dNTPs, Tris-HCl, MgCl2、(NH4)2SO4And Tween-20.
7. kit as claimed in claim 4, which is characterized in that the kit further includes negative quality-control product；And/or
The kit further includes internal standard quality-control product.
8. the method detected for hepatitis type B virus (HBV) parting and drug resistant gene, which is characterized in that the method includes steps It is rapid:
(1) sample to be detected is provided, and extracts hbv nucleic acid from the sample to be detected；
(2) step (1) is extracted to obtained viral nucleic acid, the multiple asymmetric PCR amplification of PCR reaction solution progress is added, obtained respectively Pcr amplification product；
It wherein, include primer pair group described in claim 1 in the PCR reaction solution；
(3) PCR product hybridization check
It is added in Electrochemistry gene chip after pcr amplification product is mixed with electrochemical hybridization liquid, in Electrochemistry gene chip In detected.
9. method according to claim 8, the electrochemical hybridization liquid includes signal probe group as claimed in claim 2；With/ Or
The Electrochemistry gene chip includes capture probe group as claimed in claim 3.
10. the purposes of primer pair group as described in claim 1 and signal probe group as claimed in claim 2, is used to prepare Detection kit, the detection that the detection kit is detected for hepatitis type B virus (HBV) parting and drug resistant gene.
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