CN106636466A

CN106636466A - Precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA)

Info

Publication number: CN106636466A
Application number: CN201611259032.9A
Authority: CN
Inventors: 张小勇; 刘红艳
Original assignee: Southern Hospital Southern Medical University
Current assignee: Southern Hospital Southern Medical University
Priority date: 2016-12-30
Filing date: 2016-12-30
Publication date: 2017-05-10

Abstract

The invention relates to a precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA). The precise quantification method of the HBV cccDNA comprises steps as follows: the copy number of a sample is quantitatively controlled within 10,000 copes through nucleic acid; primers and probes of cccDNA and rcDNA are designed and detected with a double-gap method; digital PCR (polymerase chain reaction) amplification is performed; analysis parameters are adjusted in digital PCR instrument analysis software, 1/2-1/3 of the average fluorescence intensity of positive particles in positive control holes is set as a positive threshold, the to-be-detected sample is analyzed and the copy number of cccDNA in the sample is calculated using software of the digital PCR instrument, and false positive caused by non-specific amplification is eliminated. The method solves the false positive interference problem of double-gap method and the PCR method for rcDNA; compared with other digital PCR detection methods for cccDNA, the method has the advantages that specific DNA enzyme is not required to treat the sample, closed pipe operation is realized, the cross-pollution risk is reduced, and the operation process is simplified.

Description

A kind of method of hepatitis b virus covalence closed cyclic DNA accurate quantification

Technical field

The present invention relates to a kind of method of DNA accurate quantifications, more particularly to a kind of hepatitis b virus covalence closed ring The method of shape DNA (cccDNA) accurate quantification.

Background technology

Covalently closed circular DNA (covalently closed circular DNA) is referred to as cccDNA, is B-type hepatitis Replicate and set up Infection Status after malicious host cells infected in the cell, the lax cyclic DNA (rcDNA) of virus enters nucleus, Formed using host DNA polymerase and topological enzyme etc. " reparation ", structural integrity, superhelix double chain DNA molecule.CccDNA is deposited It is the duplication " pond " for becoming hepatitis B virus in nucleus, it is still no at present to enter in nucleus, and can remove The medicine of cccDNA, this is also one of the reason for easily recurring after existing antiviral treatment effect on driving birds is not good and treatment.

Detection by quantitative for cccDNA is hepatitis viruss detection difficult point, because there is various homologous hepatitis B virus DNAs point Son is simultaneously deposited (cccDNA, rcDNA, ssDNA, the HBV DNA of integration), it is necessary to efficiently separate cccDNA and other kinds of virus DNA。

At present the method for detection cccDNA includes intrusion method PCR (Wong, Hepatology, 2004:40), embedding and primer method (Jun-Bin,J Virol Methods,2003:112), across rcDNA sonde method (He, Biochem Biophys Res Commun 2002；295) etc..Intrusion method sensitivity is low, about 104copies/ml, and is easily subject to viral bootlegging process The linearized double-stranded molecule interference of generation.Chimeric primers method except be similarly subjected to linearized double-stranded molecule disturb in addition to, in high abundance rcDNA Can be disturbed by false positive in sample.Although will not be disturbed by linearized double-stranded molecule across the double breach quantitative PCR methods of rcDNA, Can be because the incomplete extension products normal chain minus strand self annealing of rcDNA causes false positive to disturb in high abundance rcDNA samples.

Digital pcr carries out microdroplet process to sample, can produce 1 nanoliter of drop that is very uniform, repeating, each sample Form 20,000 drops.Nucleic acid solution after Macrodilution is dispersed in the microreactor of chip or microdroplet, each reaction The nucleic acid-templated number of device is less than or equal to 1.So after PCR cycle, there is the reactor of a nucleic acid templates Fluorescence signal will be provided, the reactor without template is just without fluorescence signal.According to relative scale and the volume of reactor, just The nucleic acid concentration of original solution can be extrapolated.Using digital pcr the thinking similar with normal PCR can be used to carry out cccDNA to determine Amount (Di, Biotechnol Lett, 2015；Chinese Patent Application No. 201410755640.3,201510055540.4), it is former Reason is similar with traditional quantitative PCR, using specific DNA enzymatic degradation rcDNA and single stranded DNA, then carries out routine with digital pcr Quantitative work.

Problem quantitative for human liver cell cccDNA at present is mainly：Because the interference of other virus forms such as rcDNA Cause cccDNA quantitatively inaccurate, the double breach methods often used on common quantitative PCR instruments can be subject to rcDNA non-specific amplifications Interference cause false positive.

Therefore, it is necessary to develop a kind of new hepatitis b virus covalence closed cyclic DNA (cccDNA) accurate quantification Method.

The content of the invention

It is an object of the invention to provide a kind of hepatitis b virus covalence closed cyclic DNA (cccDNA) accurate quantification Method.

The method of hepatitis b virus covalence closed cyclic DNA (cccDNA) accurate quantification of the present invention, including with Lower step：

A. by the copy number of nucleic acid quantification control sample within 10000 copies；

B. using the primer and probe of double breach method design detection cccDNA and rcDNA；

C. the amplification of digital P CR is carried out, using the cccDNA plasmids of concentration known as positive control；

D. after amplification terminates, analytical parameters are adjusted in digital pcr instrument analysis software, it is average with the positives microgranule of Positive control wells The 1/2～1/3 of fluorescence intensity is set to positive threshold value, then carries software analysis sample to be tested by digital P CR instrument and calculates sample CccDNA copy numbers in this, exclude the false positive caused by non-specific amplification.

According to the further feature of the method for the invention, in step A, using spectrophotometer measurement sample OD260, OD280 numerical value, calculates the DNA concentration and purity in sample, and the concentration of sample is adjusted in 1000- using TE buffer Between 10000/ microlitre.

According to the further feature of the method for the invention, in step B, amplification condition gathers with reference to used PCR Synthase description, the design of amplification cycles number is between 15-25 circulations.

Each PCR reaction is distributed to the present invention principle of different individual drops using digital pcr, by it is traditional across The double breach quantitative PCRs of rcDNA are referred in digital pcr, it is often more important that using the principle of end-point method fluoroscopic examination, utilized CccDNA plasmids specimen determines detection threshold value as internal reference, excludes the double breach methods of rcDNA because too high incomplete extension of abundance produces The false positive interference that thing normal chain minus strand self annealing is caused.The method of the invention solves the vacation sun of the double breach method PCR methods of rcDNA Property interference problem, compared with other digital pcrs detection cccDNA, sample is processed without the need for specific DNA enzyme, realize and close Pipe is operated, and reduces cross contamination risk and streamline operation.

Description of the drawings

Fig. 1 is carried out after digital pcr amplification as positive control template using cccDNA plasmids, by machine voluntarily interpretation threshold value Testing result figure.

Fig. 2 is that, to detect that sample carries out digital pcr amplification, the threshold value arranged automatically using instrument carries out calculated inspection Survey result figure.

Fig. 3 is to carry out digital pcr amplification with identical detection sample, and it is 3500 to adjust threshold value according to the result of Fig. 1, is carried out Calculated testing result figure.

Specific embodiment

Across the double breach quantitative PCR methods of rcDNA be cccDNA detection methods popular at present, by rcDNA double-strands The indentation, there both sides design primer of molecule and probe, make pcr amplification reaction terminate in indentation, there, so as to avoid rcDNA molecules It is amplified and causes false positive, and cccDNA is because without breach, PCR can be normally carried out producing positive signal.But real reaction In, always there are part rcDNA molecules its breach is repaired completely due to the reparation characteristic of PCR amplification enzymes, become cccDNA molecules Form, so as to produce false positive results.

The method of hepatitis b virus covalence closed cyclic DNA accurate quantification of the present invention, comprises the following steps：

(1) using the OD260 of spectrophotometer measurement sample, OD280 numerical value calculates the DNA concentration and purity in sample, uses The concentration of TE buffer adjustment sample controls the copy number of sample between 1000-10000/ microlitre from there through nucleic acid quantification Within 10000 copies.

(2) using the primer and probe of double breach method design detection cccDNA and rcDNA；

The primer and probe of double breach method design cccDNA, sequence is as follows：

Forward primer：CTCCCCGTCTGTGCCTTCT(SEQ ID NO:1)；

Reverse primer：GCCCCAAAGCCACCCAAG(SEQ ID NO:2)；

Probe：FAM-CGTCGCATGGARACCACCGTGAACGCC-BHQ1.

(3) digital P CR (such as PCR instrument of Bio-Rad companies) is carried out, amplification condition is polymerized with reference to used PCR Enzyme description, amplification cycles number is designed between 15-25 circulations, using the cccDNA plasmids of concentration known as positive control；

Digital pcr experiment condition：

95 degree of 10min；95 degree of 20s, 58 degree of 60s, 25 circulations.

(4) after amplification terminates, analytical parameters are adjusted in digital pcr instrument analysis software, it is positives micro- with Positive control wells The 1/2～1/3 of grain average fluorescent strength is set to positive threshold value, then carries software analysis sample to be tested by digital P CR instrument CccDNA copy numbers in sample are calculated, the false positive caused by non-specific amplification is excluded.

(5) interpretation of result：

Experiment every time is taken a hole and is expanded as positive control template using cccDNA plasmids.During analysis, by machine voluntarily first The fluorescence threshold of interpretation Positive control wells, then arranges sample with the 1/2-1/3 of Positive control wells positive sample average fluorescent strength The interpretation threshold value in this hole, then the cccDNA quantity of sample aperture is calculated by instrument.

Fig. 1 be expanded as positive control template using cccDNA plasmids after by machine voluntarily interpretation threshold value numeral PCR testing result figures.Fig. 1 shows using cccDNA plasmids and expanded as positive control template, by machine voluntarily interpretation threshold value, Differentiate negative, positive microdroplet.As shown in Figure 1, it is seen that the fluorescence intensity of positive microdroplet (top blue dot) be located at 6000-8000 it Between, by positive microdroplet average fluorescent strength 1/2-1/3 as threshold value, therefore set 3500 as sample analyses positive threshold value.

The sample that this experiment is adopted actually does not contain cccDNA, containing only rcDNA.

Fig. 2 is to carry out digital pcr amplification with above-mentioned detection sample, and the threshold value arranged automatically using instrument is calculated Testing result figure.As shown in Fig. 2 the threshold value arranged automatically using instrument is calculated, instrument sets threshold value as 1500 certainly, so as to The sample is calculated containing 30 cccDNA copies, false positive occurs.False positive results herein should be rcDNA molecules by PCR enzymes Extending the false cccDNA molecules of formation and being further used as template amplification is caused, and this is also false-positive in normal PCR test Main source.

Fig. 3 is to carry out digital pcr amplification with identical detection sample, and it is 3500 to adjust threshold value according to the result of Fig. 1, is carried out Calculated testing result figure.As shown in figure 3, it is 3500 to arrange threshold value according to the result of Fig. 1, instrument calculates the sample containing 0 Individual cccDNA copies, it is seen that the threshold value setting eliminates rcDNA false positive results from caused by extending.

Shown by above-mentioned experiment, method of the present invention efficiently solves the vacation sun of the double breach method PCR methods of rcDNA Property interference problem, compared with other digital pcrs detection cccDNA, sample is processed without the need for specific DNA enzyme, realize and close Pipe is operated, and reduces cross contamination risk and streamline operation.

Principle analysis：Digital pcr instrument is by the way that template molecule is distributed in 20000 microdroplets, when template molecule copy number During significantly lower than microdroplet number, only comprising one or without template molecule in each microdroplet, single point is individually carried out in each microdroplet The PCR reactions of subtemplate.The PCR of more than 1 is needed before being complete double-stranded DNA by PCR enzyme reparations in view of rcDNA molecules Circulation is reacted, therefore is similarly the PCR reactions of unimolecule template, and amplification rate will at least be slower than normal cccDNA molecules mould The time of one circulation of plate.The present invention is using the principle of end-point method fluoroscopic examination, by the use of cccDNA plasmid specimen as internal reference Determine detection threshold value, exclude what the double breach methods of rcDNA were caused due to the too high incomplete extension products normal chain minus strand self annealing of demeanour False positive is disturbed.

Claims

1. a kind of method of hepatitis b virus covalence closed cyclic DNA accurate quantification, it is characterised in that comprise the following steps：

2. method according to claim 1, it is characterised in that：In step A, using spectrophotometer measurement sample OD260, OD280 numerical value, calculates the DNA concentration and purity in sample, and the concentration of sample is adjusted in 1000- using TE buffer Between 10000/ microlitre.

3. method according to claim 1, it is characterised in that：In step B, amplification condition refers to used PCR Polymerase description, the design of amplification cycles number is between 15-25 circulations.