CN106498095A - A kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus - Google Patents

Abstract

The present invention provides a kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus, including：PCR reactant liquors, the PCR reactant liquors include reaction buffer, dideoxyribonucleotide triphosphate, the upstream and downstream primer for target polynucleotide amplification and the probe detected for target polynucleotide, wherein, the probe sequence for target polynucleotide detection is SEQ ID NO：3.A kind of operation of present invention offer is quick, method is easy, detection specificity is good, the 2 type kit for detecting nucleic acid of human immunodeficiency virus that sensitivity is high, detection range is wide, the probe specificity for target polynucleotide detection that this test kit is provided is higher, apply the test kit, can to unknown sample in 2 type nucleic acid of human immunodeficiency virus be used for quickly detecting, reliable experimental basis are provided for 2 type nucleic acid of diagnosing human immunodeficiency virus, can solve the problems, such as in prior art that low test kit detection efficiency, poor specificity and sensitivity are low.

Description

A kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus

Technical field

A kind of the present invention relates to field of nucleic acid detection, more particularly to 2 type nucleic acid detection reagent of human immunodeficiency virus Box.

Background technology

Human acquired immunodeficiency syndrome (AIDS, English full name：acquired immunodeficiency Syndrome), also known as acquired immune deficiency syndrome (AIDS), its cause of disease is HIV (human immunodeficiency virus) (HIV, English full name：human Immunodeficiency virus), belong to the lentiviridae of Retroviridae (Retroviridae) (Lentivirinae).HIV-1 is divided into according to its genotypic difference and HIV-2 two is large-scale, each large-scale including multiple hypotypes.Mesh That on former world, most area is popular is HIV-1, and HIV-2 is only regional popular in West Africa, West Europe.

HIV genomes are made up of two single strand positive strand RNA, comprising tri- structural genes of gag, pol, env, and tat, The controlling genes such as rev, nef, vif, vpu, vpr, vpx, and respectively contain long terminal repeat at the 5 ' ends and 3 ' ends of genome (LTR, English full name：long terminal repeat).The LTR areas of HIV contain cis-regulatory sequence, comprising promoter and enhancing Son area containing negative regulation, control the expression of viral gene.The function of each gene is as follows：

(1) gag genes can encode the polymeric precursors albumen (P55) of about 500 aminoacid compositions, be formed through protease hydrolysiss P17, P24 nucleoprotein, makes RNA not by extraneous nucleases.(2) pol gene codes polymerase precursor protein (P34), through cutting Cut to form protease, intergrase, reverse transcriptase, ribonuclease H, be virus multiplication institute required.(3) env gene codes are about Precursor protein glycosyl chemical conversion gp160, gp120 and the gp41 of 863 aminoacid.Gp120 contains neutralization antigenic determinant, demonstrate,proves Bright HIV Neutralization and crystallizations, on gp120V3 rings, V3 ring regions are the critical function areas of envelope protein, in virus and cell fusion In play an important role.Gp120 is connected with non-covalent bond with transmembrane protein gp41.Gp41 is merged with target cell, promotes cell entry Intracellular.Experiment shows that gp41 also has compared with strong antigen, can inducing antibodies reaction.(4) tat gene coded proteins (P14) Can be combined with LTR, to increase viral all genetic transcription rates, also can be promoted the translation of virus mRNA after transcription.(5) rev bases Because product is a kind of cis-activating factor, sequence (Crs, English full name can be suppressed to cis acting in env and gag：Cis- Acting repression sequance) effect of disinthibiting, strengthen the expression of gag and env genes, to synthesize corresponding virus Structural protein.(6) nef gene coded proteins P27 has negative regulation to act on to the expression of HIV genes, to postpone virus replication.The egg Act on the LTR of HIV cDNA in vain, suppress the virus transcription that integrates.Possibly HIV maintains persistent infection institute required in vivo. (7) vif gene pairss HIV is not essential, but may affect free HIV, the generation of virion and propagate in vivo. (8) vpu genes are peculiar for HIV-1, indispensable with maturation to effective duplication of HIV and the assembling of virion.(9) vpr genes Encoding proteins are a kind of weak transcriptional activators, are played a role in vivo in the breeding cycle.HIV-2 gene structures and HIV-1 Difference is there are, homology is only that 40～60%, HIV-2 does not contain vpu genes between the two, but there are the not clear vpx genes of a function. , because its reverse transcription is without calibration function, mispairing is high, causes genetic variability very strong, especially with env genovariations rate most for HIV High.According to the mutation of env gene regions, HIV-2 can be divided into 7 hypotypes again.

Quantitative fluorescent PCR (polymerase chain reaction, English full name：Polymerase Chain Reaction) it is general Fluorophor is added on the basis of logical PCR in PCR reaction systems, during sequence amplification, with PCR product not Disconnected accumulation, also equal proportion increases fluorescence signal intensity, and fluorescence signal constantly strengthens, and builds up to form an amplification curve, By the whole PCR processes of fluorescence cumulative curve real-time monitoring, unknown template is carried out finally by standard curve qualitative and quantitative The method of analysis.

The at present fluorescence chemical method used by real-time Q-PCR mainly has five kinds, is respectively：DNA combines dyeing, Hydrolysis probes, molecular beacon, fluorescent dye primer, hybridization probe.They can be divided into the special and non-specific inspection of extension increasing sequence again Survey two big class.

(1) the non-specific detection of extension increasing sequence.The method is based on the fluorescence molecule combined with DNA, such as SYBR green 1 Deng fluorescent dye.By 1 fluorescent dyes of excess SYBR green, this dyestuff is added in PCR reaction systems can penetrate into DNA double Chain, and fluorescence signal is sent, but can not be combined with single stranded DNA or RNA.During template amplification, with double-stranded DNA quantity Accumulation, fluorescence signal intensity are also waited than strengthening.The advantage of fluorescent dye is that it can monitor the amplification of any dsDNA sequences, no The design of probe is needed, makes detection method become easy, while also reducing the cost of detection.But just because of fluorescent dye Can combine with any dsDNA, therefore it also can be combined with non-specific dsDNA (such as primer dimer), experiment is easily produced False positive signal.The problem of primer dimer can be solved with the software with melting curve analysis at present.

(2) extension increasing sequence specific detection.The nineties in last century, 5 ' exonuclease activities of Taq DNA polymerases It was found that and double fluorescence labeling probe with causing to be possibly realized in a closed PCR reaction tubes middle ground detection PCR primer, this Afterwards, the commercialization development of corresponding instrument and reagent causes real-time Q-PCR methods formal input in research work to make With.Extension increasing sequence specific detection is divided into direct method and indirect method again.

Directly method refers to the probe of mark fluorescent and amplified production is combined rear i.e. direct generation fluorescence, such as molecule letter Mark, fluorescent dye primer and molecular probe probe.Molecular beacon probe is a kind of hairpin probe of mark fluorescent, works as probe molecule During in hairpin structure, in conjunction with close in the fluorophor at its two ends distance so that produce energy transfer effect, and do not occur glimmering Light.In the presence of having the template complementary with ring sequence to deposit sequence, ring sequence will be matched with template, and molecular beacon will be glimmering into chain Light group and quencher separate, and send fluorescence signal.Fluorescent dye primer is produced from the concept change of molecular beacon Joint molecular probe system is planted, it is directly combined the sequence of the hairpin structure of fluorophor labelling with PCR primer, so that Fluorescence marker groups are directly incorporated in pcr amplification product.Mainly there are two kinds at present：Sunrise primer and scorpions.Hybridization probe Using two special probes, 3 ' ends of the probe of its middle and upper reaches are marked with donor fluorescent element, and the 5 ' of the probe in downstream ends are marked Note has acceptor fluorescence element.Template annealing stage in PCR, two probes simultaneously hybridized with amplified production, and formed and combined end to end Form, make donor and acceptor fluorescence element distance closely, both produce FRET (fluorescence resonance energy transfer) (FRET, this effect with upper The mode for stating hydrolysis probes is contrary) so that acceptor fluorophore sends fluorescence；When two probes are in free state, unstressed configuration Produce.As two probes have been used in reaction, therefore increased the specificity of method, be in addition possible with fluorogenic oligonucleotides The sequence combined with oligonucleotide probe by melting curve pair is analyzed, and therefrom obtains useful information.

Round-about way is exactly the strategy using hydrolysis probes.Most widely used in real-time Q-PCR at present TaqMan systems are exactly to have used this principle.When PCR is expanded, while pair of primers is added, addition one is specific glimmering Light probe, the probe is an oligonucleotide, two ends difference one reporter fluorescence group of a labelling and quenching fluorescence group, now After 5 ' end fluorophor energy absorptions (there is fluorescence resonance energy to turn in energy transfer to the 3 ' end fluorescent quenching groups that closes on Move, FRET), when therefore probe is complete, can't detect the fluorescence that the end of the probe 5 ' fluorophor sends.But it is in PCR amplifications, molten After template denaturation in liquid during process annealing, primer is combined with template simultaneously with probe.Under the mediation of primer, along template forward Probe junction is extended to, the displacement that chain occurs, (this activity is double-stranded specific to 5 ' -3 ' 5 prime excision enzyme activity of Taq enzyme, trip From single-stranded probe unaffected) by probe 5 ' hold connection fluorophor cut down from probe, be free on reaction system In, so as to depart from the shielding of 3 ' end fluorescent quenching groups, receive polished bard and inspire fluorescence signal, i.e., often expand a DNA, just There is a fluorescence molecule to be formed, it is achieved that the accumulation of fluorescence signal forms Complete Synchronization with PCR primer.

Although HIV-2 is regional infection, to the west of based on the area of non-sum West Europe, add recently as movement of population By force, cases of infection are also occurred in that in other areas.HIV-2 aggressivity is weaker compared with HIV-1, develops into the incubation period of acquired immune deficiency syndrome (AIDS) Longer, but later stage symptom is similar, and hazardness is suitable.Traditional Serologic detection such as ELISA method, immunofluorescence, agglutination test, Immunoblotting (WB, English full name：Western blot) HIV-2 can not be detected in infection window phase, and HIV-2 with There is certain cross reaction in HIV-1 in serology, easily cause missing inspection or flase drop, make troubles to anaphase and medication. And although regular-PCR is time-consuming less, as HIV-2 variability is strong, hypotype is more, causes specificity poor, and its later stage Interpretation of result need to be uncapped to be carried out, and easily causes pollution and non-specific amplification causes false positive, increased the difficulty of detection.Therefore, Need a kind of sensitivity badly high, high specificity, broad covered area, detection window phase short HIV-2 detection methods.

Content of the invention

The present invention provides a kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus, to solve prior art pilot scale Agent box sensitivity is low, the technical problem of poor specificity.

The present invention provides a kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus, the human immunodeficiency Viral 2 type kit for detecting nucleic acid include：2 type nucleic acid PCR reactant liquor of human immunodeficiency virus, the PCR reactant liquors bag Include reaction buffer, dideoxyribonucleotide triphosphate, the upstream and downstream primer for target polynucleotide amplification and be used for target multinuclear The probe of thuja acid detection, wherein, the probe sequence for target polynucleotide detection is SEQ ID NO：3.

Preferably, the sequence of the upstream and downstream primer for target polynucleotide amplification is respectively SEQ ID NO：1 and 2.

Preferably, 2 type kit for detecting nucleic acid of the human immunodeficiency virus also includes internal standard, the interior target sequence It is classified as SEQ ID NO：7.

Preferably, 2 type kit for detecting nucleic acid of the human immunodeficiency virus also includes：For internal standard fragment amplification Upstream and downstream primer and for detecting interior target probe, the sequence of the probe is SEQ ID NO：6.

Preferably, the upstream and downstream primer sequence for internal standard fragment amplification is respectively SEQ ID NO：4 and 5.

Preferably, the detection kit also includes positive control and negative control.

Preferably, also include enzyme mixation in the detection kit, the enzyme mixation includes hot resistant DNA polymerase And uracil dna glycosylase, while also include dUTP in the PCR reactant liquors.

Nucleotide sequence in the present invention is as shown in table 1：

The technical scheme that embodiments of the invention are provided can include following beneficial effect：

The present invention provides a kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus, including：Human immunodeficiency Viral 2 type nucleic acid PCR reactant liquors, the PCR reactant liquors include reaction buffer, dideoxyribonucleotide triphosphate, many for target The upstream and downstream primer of amplification oligonucleotide and the probe for target polynucleotide detection, wherein, described for target polynucleotide inspection The probe sequence of survey is SEQ ID NO：3.The present invention provides that a kind of operation is quick, method is easy, detection specificity is good, sensitivity The wide 2 type kit for detecting nucleic acid of human immunodeficiency virus of high, detection range, this test kit provide for many nucleoside of target Acid detection probe specificity higher, apply the test kit, can to unknown sample in 2 type core of human immunodeficiency virus Acid is used for quickly detecting, and provides reliable experimental basis for 2 type nucleic acid of diagnosing human immunodeficiency virus, can solve existing Technology pilot agent box detection efficiency is low, the problem that poor specificity and sensitivity are low.

It should be appreciated that above general description and detailed description hereinafter are only exemplary and explanatory, not The present invention can be limited.

Specific embodiment

Each embodiment in this specification is described by the way of going forward one by one, identical similar portion between each embodiment Divide mutually referring to what each embodiment was stressed is the difference with other embodiments.

Embodiment one

The present invention provides a kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus, the human immunodeficiency Viral 2 type kit for detecting nucleic acid include：2 type nucleic acid PCR reactant liquor of human immunodeficiency virus, the PCR reactant liquors bag Include：30 μ l reaction buffers, 1.5 μ l of dideoxyribonucleotide triphosphate, the 0.5 μ l of 100mM are used for the spy of target polynucleotide detection Pin, its nucleotides sequence are classified as SEQ ID NO：3 and the upstream and downstream primer for target polynucleotide amplification, its nucleotide sequence point Wei not SEQ ID NO：1 and 2.

The genomic homology of HIV-2 and HIV-1 is relatively low, only 40%-60%, and mutability is strong.For HIV-2 genes Design a pair of upstream and downstream primers and TaqMan probe (SEQ ID NO in the LTR genes high conserved region of group 5 ' and 3 ':1st, 2,3), probe 5 ' and 3 ' respectively flag Fs AM and BHQ1, can realize detecting the high coverage rate of each hypotypes of HIV-2, at the same can by its with HIV-1 and Other viruses make a distinction, with higher specificity.

The present invention provides a kind of quick operation, method simplicity, detection specificity is good, sensitivity is high, the people that detection range is wide 2 type kit for detecting nucleic acid of para-immunity defective viruss, the probe for target polynucleotide detection that this test kit is provided are special Property is higher, applies the test kit, can to unknown sample in 2 type nucleic acid of human immunodeficiency virus be used for quickly detecting, Reliable experimental basis are provided for 2 type nucleic acid of diagnosing human immunodeficiency virus, test kit inspection in prior art can be solved Survey the problem that efficiency is low, poor specificity and sensitivity are low.

Embodiment two

The present invention provides a kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus, the human immunodeficiency Viral 2 type kit for detecting nucleic acid include：2 type nucleic acid PCR reactant liquor of human immunodeficiency virus, the PCR reactant liquors bag Include：30 μ l reaction buffers, 1.5 μ l of dideoxyribonucleotide triphosphate, the 0.5 μ l of 100mM are used for the spy of target polynucleotide detection Pin, its nucleotides sequence are classified as SEQ ID NO：3rd, it is used for the upstream and downstream primer of target polynucleotide amplification, its nucleotide sequence difference For SEQ ID NO：1 and 2, internal standard, its sequence is SEQ ID NO：7th, it is used for the upstream and downstream primer of internal standard fragment amplification and is used for Target probe in detection, its sequence are respectively SEQ ID NO：4th, 5 and 6.

Design a kind of synthetic by coat protein wrap up containing specific RNA sequence (SEQ ID NO：7) slow viruss As internal standard Quality Control, primer (the SEQ ID NO of a pair of detection internal standard Quality Control sequences of another design：4th, 5) and a TaqMan probe (SEQ ID NO：6), the difference of probe 5 ' and 3 ' labelling HEX and BHQ1, are monitored to the extraction and amplification procedure of target sequence, keep away Exempt from false negative.

Additionally, in the present embodiment, also include the metal cation required for Tth enzymes, Taq enzyme and catalytic dna polymerase, In the present embodiment, the proportioning of PCR reaction systems is as shown in table 2：

Table 2：The proportioning of PCR reaction systems

Component	Volume in each reaction
		Reaction buffer	30ul
dNTPs(100mM)	1.5μl
		Tth enzymes (5U/ μ l)	2.0μl
Taq enzyme (5U/ μ l)	2.0μl
		Mn(OAc)2(30mM)	1.5μl
SEQ ID NO:1	0.5μl
		SEQ ID NO:2	0.5μl
SEQ ID NO:3	0.25μl
		SEQ ID NO:4	0.3μl
SEQ ID NO:5	0.3μl
		SEQ ID NO:6	0.15μl
Sterilizing purified water	Up to 50μl

Additionally, in other embodiments of the invention, also including negative control and positive control.The present invention is made using negative serum For negative control, using synthetic finite concentration (1 × 103copies/ml) pseudoviruss as positive control.

Method provided in the present invention is a kind of real-time fluorescence reverse transcription PCR (Real time RT-PCR), more specifically Say as a kind of real-time fluorescence reverse transcription PCR of extension increasing sequence specific detection on ground.One section of two ends is introduced in regular-PCR system The TaqMan probe with nucleic acid-templated complementation with fluorescent chemical group (reporter fluorescence group and quenching fluorescence group).

Primer used by present invention detection HIV-2 is the oligonucleoside for synthesizing starting point as HIV-2 nucleotide sequences amplifying nucleic acid Acid (SEQ ID NO:1、2).Primer used by detection HIV-2 internal standard Quality Controls is closed for can serve as internal standard Quality Control nucleotide sequence amplifying nucleic acid Oligonucleotide (SEQ ID NO into starting point:4、5).Using conventional method from restrictive digestion content purification obtain above-mentioned Primer, or above-mentioned primer can be produced by synthetic method.Primer preferably efficiency highest in amplification is single-stranded, but primer It can be double-strand.Double-chain primer is denatured (such as making its degeneration using heating means) first, that is, process to separate each chain.At this In bright, a fluorescently-labeled TaqMan probe (SEQ ID NO is respectively devised for HIV-2 target nucleotides and internal standard RNA：3、 6), wherein, oligonucleotide probe SEQ ID NO：3 two ends are respectively labeled as FAM and BHQ1, oligonucleotide probe SEQ ID NO：6 two ends are respectively labeled as HEX and BHQ1.When amplification starts, probe is specific with the complementary decision of HIV-2 target nucleic acid sequences Hybridize with template sequence on the ad-hoc location that position or internal standard Quality Control nucleic acid array complementation determine, circumscribed in polymerase 5 ' -3 ' It is hydrolyzed under enzymatic activity, discharges fluorescence signal.

First, catalysis of the HIV-2 nucleotide sequences through reverse transcriptase, completes the cDNA synthesis under RNA is instructed, subsequent DNA Become cDNA through heat denatured (about 90-105 DEG C of denaturation temperature general range, the time typically carries out about 30 seconds or more long). When amplification starts, probe is combined with template, and subsequently, 5 ' ends -3 ' of Taq enzyme hold 5 prime excision enzyme activity that probe enzyme action is degraded, and make Reporter fluorescence group is separated with quenching fluorescence group, so as to fluorescence monitoring system can receive fluorescence signal.With entering for reaction OK, product is constantly accumulated, and also equal proportion increases fluorescence signal intensity.Often circulate through one, collect a fluorescence intensity Signal, the change for monitoring product amount by fluorescence intensity change, obtains a fluorescent amplification curve figure.Fluorescent amplification curve can be with It is divided into three phases：Fluorescence background stage, fluorescence signal exponential amplification stage and plateau.In fluorescence background rank Section, the level for producing fluorescence can not be significantly distinguished with background, during exponential phase, between the logarithm value of product amount and starting template amount There is linear relationship, in plateau, amplified production increase no longer exponentially therefore can be in reaction in exponential phase The amount of product, and thus initial to infer template content is detected on certain point.

The detection method of the present invention is Real time RT-PCR, and RNA reverse transcriptions and DNA cloning are combined.Typically Real time RT-PCR courses of reaction be：1) cDNA synthesis；2) cDNA denaturations, time and length depend on target nucleotide Length and base composition, generally 90 DEG C -105 DEG C of the temperature of denaturation, the time is generally 1-10min, and the purpose of denaturation is It is completely separated Double-stranded nucleotide sequence for single-stranded；3) degeneration, generally 90 DEG C -105 DEG C of temperature, the time is generally 10s-30s； 4) anneal, make on the target sequence of each primer annealing to HIV-2 or internal standard Quality Control nucleic acid.The temperature of annealing is usually 40 DEG C -60 DEG C, The time of annealing can be 10s-60s；5) extend, primer is combined with template, starts to synthesize new double-stranded DNA, elongating temperature one As be 40 DEG C -80 DEG C, extension of time can be 10s-5min.

Fluorescence detection channel is selected：1) FAM passage (Reporter are selected:FAM,Quencher:None) the detection mankind exempt from 2 type nucleic acid of epidemic disease defective viruss；2) HEX passage (Reporter are selected:ROX,Quencher:None) internal standard is detected；4) reference Fluorescence (Passive Reference) is set to none.Fluorescent quantitation real-time PCR reactions condition is as shown in table 3.

Table 3：Fluorescent quantitation real-time PCR reactions condition

Interpretation of result：

After reaction terminates, instrument automatically saves result, it is possible to use the software that instrument is carried is automatically analyzed (can also Initial value, end value and the threshold line value for adjusting baseline manually is analyzed), then record sample Ct values and definite value result. Amplification curve and the intersection point of threshold line, (i.e. cycle threshold refer to that the fluorescence signal in PCR reaction tubes reaches and set referred to as Ct The cycling numerical value experienced during fixed threshold value).Concrete test result analysis are as follows：

1) when FAM passages are without CT values, and during HEX channel Cs T value 40 (generally 28-32), can report that testing result is the moon Property；

2) when FAM channel Cs T value 40, and HEX channel Cs T value 40, can report that testing result is the positive；

3) when FAM channel Cs T value 40, and HEX channel Cs T value 40, can report that concentration of specimens is offline less than detection, as a result Only for reference；

4) when HEX channel Cs T value 40, the testing result is invalid, should search and exclude reason, and retrial of laying equal stress on is tested；

5) when occur negative control have CT values or in typical case's S type amplification curves, positive control without CT values or without amplification curve During any one in two kinds of situations, the testing result is invalid, should search and exclude reason, and retrial of laying equal stress on is tested.

For proving the feasibility of the inventive method, tested as follows：

(1) lowest detectable limit (LOD) test

By detecting to the sample of each Concentraton gradient, show that the detection sensitivity (LOD) of this detection method is 15IU/ mL.

(2) the cross reaction situation of specificity experiments and Other diseases

By being detected to other pathogen beyond HIV-2 with the detection method in the present invention, as a result show this Method in bright is to pathogenic infection samples such as 3 group types of O, M, N of HIV-1,8 types of HBV A-H, HCV1-6 types, Epstein-Barr virus No cross reaction, shows which has high Non-specific.

(3) anti-interference to potential endogenous substance

Test shows, as triglyceride 3000mg/dL in plasma sample, non binding bilirubin 40mg/dL, blood red When albumen 500mg/dL, haemproteins 9g/dL, the sensitivity of this detection method is interference-free.When hemocyte in plasma sample During volume ratio 2.5, it is possible that the phenomenon of internal standard failure.

(4) anti-interference to potential allogenic material

Test shows, when containing Zidovudine, tenofovir, lamivudine, Lopinavir Li Tuonawei, A De in sample The common antiviral such as good fortune Wei ester, Sebivo, Entecavir, tenofovir disoproxil, acetaminophen, zanamivir (200 μ g/ml) During medicine, the detection sensitivity of this test kit will not be substantially interfered with.

Detection method in the present invention is combined together PCR, fluorescent probe, is made PCR based on real-time fluorescence reverse transcription PCR The overall process of amplification and product analysis is carried out under single tube sealing condition, can carry out real-time monitoring to course of reaction.Glimmering in real time Light quantitative technique is a kind of nucleic acid detection technique for quickly growing in recent years, using a kind of amplification with fluorescence detection device Instrument, fluorescence detection device can send the exciting light of specific wavelength according to certain routines periodically, collect detection fluorescence letter Number, the level of amplification of each circulation of the dynamic change reflection in real time by detecting fluorescence signal, according to amplification curve and CT values May determine that the yin and yang attribute of testing result.Meanwhile, it is used as qualitative reference product by designing the false positive sample of variable concentrations, can leads to Cross the absolute concentration that standard curve calculates sample.

Being had the advantage that compared with other detection techniques with real-time fluorescence PCR technology for detection HIV-2 in the present invention：

(1) compared with traditional immunoassay using real-time fluorescence PCR technology for detection HIV-2, with higher Sensitivity, as long as theoretically there is a gene copy just detect, while avoiding cross reaction, shortens inspection Survey window phase.

(2) according to the unique conserved genetic sequences design primer of HIV-2 genomes and probe, PCR reactions ensure that High degree of specificity.The genomic homology of HIV-2 and HIV-1 is relatively low, only 40%-60%, and mutability is strong, and is located at gene The env genes of group zone line are more very.The present invention avoids high Sudden change region in design of primers, for the 5 ' of HIV-2 genomes Pair of primers probe is designed in the LTR of LTR and 3 ' high conserved regions, can realize that the high coverage rate to each hypotypes of HIV-2 is detected, while can be by Itself and HIV-1 and other viruses make a distinction, with higher specificity.

(3) slow viruss using synthetic are used as internal standard Quality Control, it is ensured that the accuracy of testing result.Adopt in the present invention A kind of wrapped up by protein coat, used as internal standard Quality Control, internal standard Quality Control may participate in the lentiviral particle containing specific RNA sequence The extraction of HIV-2 nucleic acid and PCR reactions, monitoring experiment operating process and PCR courses of reaction, it is to avoid false negative occur, increased The credibility of testing result.

(4) integrate the high sensitivity of PCR and the two big advantage of high specific of TaqMan probe, change to a great extent The defect of normal PCR is become, has shortened detection time, simplified detecting step, improve detection efficiency.

(5) detection overall process is carried out under single tube sealing condition, it is to avoid due to intersecting the false positive for causing between sample And environmental pollution.

(6) can continuously detect the change of live signal during PCR, it is to avoid normal PCR " plateau, imitates Should ", and the quantitative of template does not pass through end-product, and calculated by Ct values, accuracy and susceptiveness are all improved.

Invention described above embodiment, does not constitute limiting the scope of the present invention.Any in the present invention Spirit and principle within modification, equivalent and the improvement made etc., should be included within the scope of the present invention.

The above is only the specific embodiment of the present invention, makes skilled artisans appreciate that or realizing this Bright.Multiple modifications of these embodiments will be apparent to one skilled in the art, as defined herein General Principle can be realized without departing from the spirit or scope of the present invention in other embodiments.Therefore, the present invention The embodiments shown herein is not intended to be limited to, and is to fit to and principles disclosed herein and features of novelty phase one The most wide scope for causing.

SEQUENCE LISTING

<110>Hunan Shengxiang Biological Technology Co., Ltd.

<120>A kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus

<130> 2016

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 21

<212> DNA

<213>Primer

<400> 1

gcaggtagag cctgggtgtt c 21

<210> 2

<211> 22

<212> DNA

<213>Primer

<400> 2

gaagagggct ttaagcaagc aa 22

<210> 3

<211> 27

<212> DNA

<213>Probe

<400> 3

tgctagactc tcaccagyrc ttggccg 27

<210> 4

<211> 22

<212> DNA

<213>Primer

<400> 4

taggaggtag agcctgggtg tt 22

<210> 5

<211> 17

<212> DNA

<213>Primer

<400> 5

ccagcaccgg ccaagtg 17

<210> 6

<211> 28

<212> DNA

<213>Probe

<400> 6

agcctgggtg ttccctgcta gactctca 28

<210> 7

<211> 154

<212> DNA

<213>Internal standard

<400> 7

tagattagga ggtagagcct gggtgttgtc acaacatttg tgttgtaagt gtaataatca 60

acttcagcta gtagtagaaa cctcgcaaga cagcctgggt gttccctgct agactctcat 120

ggacacacta cacttggccg gtgctggtgt gttt 154

Claims (7)

1. a kind of 2 type kit for detecting nucleic acid of human immunodeficiency virus, it is characterised in that human immunodeficiency's disease Malicious 2 type kit for detecting nucleic acid include：2 type nucleic acid PCR reactant liquor of human immunodeficiency virus, the PCR reactant liquors include Reaction buffer, dideoxyribonucleotide triphosphate, for target polynucleotide amplification upstream and downstream primer and be used for many nucleoside of target The probe of acid detection, wherein, the probe sequence for target polynucleotide detection is SEQ ID NO：3.

2. 2 type kit for detecting nucleic acid of human immunodeficiency virus according to claim 1, it is characterised in that described Sequence for the upstream and downstream primer of target polynucleotide amplification is respectively SEQ ID NO：1 and 2.

3. 2 type kit for detecting nucleic acid of human immunodeficiency virus according to claim 1, it is characterised in that described 2 type kit for detecting nucleic acid of human immunodeficiency virus also includes internal standard, and the interior target sequence is SEQ ID NO：7.

4. 2 type kit for detecting nucleic acid of human immunodeficiency virus according to claim 3, it is characterised in that described 2 type kit for detecting nucleic acid of human immunodeficiency virus also includes：Upstream and downstream primer for internal standard fragment amplification and it is used for Target probe in detection, the sequence of the probe is SEQ ID NO：6.

5. 2 type kit for detecting nucleic acid of human immunodeficiency virus according to claim 4, it is characterised in that described Upstream and downstream primer sequence for internal standard fragment amplification is respectively SEQ ID NO：4 and 5.

6. the 2 type kit for detecting nucleic acid of human immunodeficiency virus according to any one of claim 1-5, its feature exist In the detection kit also includes positive control and negative control.

7. 2 type kit for detecting nucleic acid of human immunodeficiency virus according to claim 6, it is characterised in that described Also include enzyme mixation in detection kit, the enzyme mixation includes hot resistant DNA polymerase and Uracil DNA Glycosylase Enzyme, while also include dUTP in the PCR reactant liquors.