CN104911255B - For detecting primer pair and probe and its application for the treatment of AIDS medicine DDI and TDF resistant mutational site - Google Patents
For detecting primer pair and probe and its application for the treatment of AIDS medicine DDI and TDF resistant mutational site Download PDFInfo
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Abstract
The invention provides the primer pair and probe for detecting treating AIDS medicine DDI and TDF resistant mutational site, includes detecting the 65th, 70 and 151 mutational site K65R, K70E and Q151M of the pol genes of the viral RNAs of HIV 1 ARMS primer pairs and Taqman probes.Present invention also offers the application of above-mentioned primer pair and probe.The present invention is compared with traditional sequencing methods, detection sensitivity is high, specificity is good, testing cost is low, medication guide is provided for Clinical HIV patient treatment, realize AIDS patient's individualized treatment, AIDS patient's medication validity can be reacted in time, improve the life quality of AIDS patient, be with a wide range of applications and long-range social benefit.
Description
Technical field
The invention belongs to biomedicine field, is related to a kind of for detecting treating AIDS medicine DDI and TDF medicament-resistant mutation
The primer pair and probe in site, above-mentioned primer pair and probe are further related in detection treating AIDS medicine DDI and TDF medicament-resistant mutation
Application in site.
Technical background
Most of popular AIDS is as caused by HIV-1 in the world.HIV-1 genomes include gag, pol and env tri-
6 regulatory gene of individual structural gene and tat, rev, nef, vpr, vif, vpu, it is long repetitive sequence in 5 ' ends and 3 ' ends
(LTR).Pol gene coded proteins enzyme, integrase and reverse transcriptase, these three viral major protein enzymes be used for amyloid protein precursor into
The integration of ripe, virus genome RNA reverse transcription and viral cDNA on host genome DNA.
More than the 30 kind anti-HIV-1 medicines that circulated in the market are used for the clinical treatment of AIDS.Due to HIV-1 reverse
Record enzyme replicated again rapidly without calibration function in a replication process so that HIV-1 be easy to evolution, genetic diversity it is extensive,
Prerequisite is provided for HIV-1 rapid produce of medicament-resistant mutation.At present, corresponding to the HIV-1 curatives of every kind of Clinical practice
Thing has the generation of its corresponding medicament-resistant mutation.
Didanosine（DDI）And tenofovir disoproxil fumarate（TDF）It is currently used two kinds of HIV nucleoside reverse transcriptases
Inhibitor (NRTIs), but because the generation of medicament-resistant mutation greatly reduces the validity of both medicines.Study channel syndrome
It is bright to cause tri- kinds of mutation of K65R, K70E and Q151M that main medicament-resistant mutation caused by DDI and TDF resistances is pol structural genes.
The method for being applied to detection HIV-1 medicament-resistant mutations at this stage is mainly PCR primer direct sequencing.Due to this method
Detection cycle is longer, somewhat expensive, non-close operation, it is difficult to cross pollution is avoided, and due to existing for DNA direct Sequencings
Sensitivity is relatively low, the problems such as heterozygous mutant so that it is difficult to obtain accurate data by once sequencing, it is necessary to which sequencing is repeated several times
The problems such as being only possible to avoid false positive, therefore direct Sequencing method is difficult to be applied to clinical detection.
With the increasingly exacerbation of resistance situation during treating AIDS, clinically there is an urgent need to develop can quickly,
Accurately, HIV-1 medicament-resistant mutation detection methods that are easy to operate and avoiding cross pollution, for meeting clinical application and checkout and diagnosis
Requirement in terms of the ageing and Personalized medicine of aspect.
The content of the invention
It is an object of the present invention to detect treating AIDS medicine DDI and TDF resistant mutational site to solve prior art
Sensitivity existing for method is low, poor specificity, cumbersome, testing cost is high, spend the time it is long the problems such as, the invention provides
A kind of high sensitivity, specificity is good, testing cost is low, quick, simple treating AIDS medicine DDI and TDF medicament-resistant mutation position
The fluorescent quantificationally PCR detecting kit of point, additionally provides a kind of application method of detection kit.
Technical scheme：The invention provides the primer for detecting treating AIDS medicine DDI and TDF resistant mutational site
Pair and probe, including the 65th, 70 of pol genes of detection HIV-1 viral RNAs and 151 mutational sites K65R, K70E and
Q151M ARMS primer pairs and Taqman probes；
The sense primer and anti-sense primer of K65R ARMS primer pairs are respectively SEQ ID No.1 and SEQ ID No.2 institutes
The nucleotide sequence shown, Taqman probes are the nucleotide sequence shown in SEQ ID No.3, respectively with FAM and BHQ1 marks 5 '
End and 3 ' ends；
The sense primer and anti-sense primer of K70E ARMS primer pairs are respectively SEQ ID No.4 and SEQ ID No.2 institutes
The nucleotide sequence shown, Taqman probes are the nucleotide sequence shown in SEQ ID No.3, respectively with HEX and BHQ1 marks 5 '
End and 3 ' ends；
The sense primer and anti-sense primer of Q151M ARMS primer pairs are respectively SEQ ID No.5 and SEQ ID No.6 institutes
The nucleotide sequence shown, Taqman probes are the nucleotide sequence shown in SEQ ID No.7, respectively with ROX and BHQ1 marks 5 '
End and 3 ' ends.
Present invention also offers another primer for being used to detect treating AIDS medicine DDI and TDF resistant mutational site
Pair and probe, including detection HIV-1 viral RNAs pol genes the 65th mutational site K65R ARMS primer pairs and
Taqman probes；
The sense primer and anti-sense primer of K65R ARMS primer pairs are respectively SEQ ID No.1 and SEQ ID No.2 institutes
The nucleotide sequence shown；
The Taqman probes of ARMS primer pairs are the nucleotide sequence shown in SEQ ID No.3, are marked respectively with FAM and BHQ1
The end of note 5 ' and 3 ' ends.
Present invention also offers another primer for being used to detect treating AIDS medicine DDI and TDF resistant mutational site
Pair and probe, include detection HIV-1 viral RNAs pol genes the 70th mutational site K70E ARMS primers and Taqman
Probe；
The sense primer and anti-sense primer of K70E ARMS primer pairs are respectively SEQ ID No.4 and SEQ ID No.2 institutes
The nucleotide sequence shown；
The Taqman probes of ARMS primer pairs are the nucleotide sequence shown in SEQ ID No.3, are marked respectively with HEX and BHQ1
The end of note 5 ' and 3 ' ends.
Present invention also offers another primer for being used to detect treating AIDS medicine DDI and TDF resistant mutational site
Pair and probe, including detection HIV-1 viral RNAs pol genes the 151st mutational site Q151M ARMS primers and
Taqman probes；
Shown in sense primer and anti-sense primer difference SEQ ID No.5 and SEQ the ID No.6 of Q151M ARMS primer pairs
Nucleotide sequence；
The Taqman probes of ARMS primer pairs are the nucleotide sequence shown in SEQ ID No.7, are marked respectively with ROX and BHQ1
The end of note 5 ' and 3 ' ends.
Present invention also offers above-mentioned primer pair and probe in detection treating AIDS medicine DDI and TDF medicament-resistant mutation position
Application in point.
It is used to detect the kit for the treatment of AIDS medicine DDI and TDF resistant mutational site present invention also offers a kind of,
Including preceding claim ARMS primer pairs and probe.
Preferably, it is right also to include RT-PCR mixed liquors, mixed enzyme solution, positive reference substance mixed liquor, feminine gender for the kit
According to savoring mixed liquor；The RT-PCR mixed liquors include RT-PCR buffer solutions, dNTPs, MgCl2, 3 pairs of upstream and downstream primers and 3 spies
Pin；The mixed enzyme solution includes M-MLV reverse transcriptases and Taq archaeal dna polymerases.
It is highly preferred that each component content is respectively in the kit：
RT-PCR mixed liquors 875uL；
The uL of mixed enzyme solution 125；
The uL of positive quality control product 45；
The uL of positive reference substance mixed liquor 45；
The uL of negative controls mixed liquor 45.
It is highly preferred that in the kit：
The RT-PCR mixed liquors include：
RT-PCR buffer solutions（20mM Tris-Hcl, 100mM NaCl, pH8.3）Final concentration 1 ×；
DNTPs final concentrations 3mM；
MgCl2Final concentration of 1.6mM；
The final concentration of the upstream and downstream primer of each ARMS primer pairs is 200nM；The Taqman of each ARMS primer pairs and examination
The Taqman probe final concentrations of agent box quality-control product primer pair are 300nM；
The mixed enzyme solution includes：
M-MLV reverse transcriptase final concentration 2U/ μ L；
Taq DNA polymerase final concentration 0.05U/ μ L；
Template cDNA final concentration 0.1~10ng/ μ L in the positive reference substance mixed liquor.
Present invention also offers mentioned reagent box in treating AIDS medicine DDI and TDF resistant mutational site is detected
Using comprising the following steps：
（1）Total RNAs extraction：The RNA of sample to be tested is extracted, mentions template ribonucleic acid solution；
（2）Reactive component is prepared：Detection mixture, positive reference substance mixture and negative controls mixing are prepared respectively
Thing；Wherein, detect mixture and prepare a person-portion by PCR mixed liquors 17.5uL, mixed enzyme solution 2.5uL, template ribonucleic acid solution 5uL；Sun
Property reference substance mixture prepare a person-portion by PCR mixed liquors 17.5uL, mixed enzyme solution 2.5uL, positive reference substance mixed liquor 5uL；
Negative controls mixture prepares a people by PCR mixed liquors 17.5uL, mixed enzyme solution 2.5uL, negative controls mixed liquor 5uL
Part；
（3）Amplification：Respectively will detection mixture, positive quality control product mixture, positive reference substance mixture and negative control
Product mixture is put into quantitative real time PCR Instrument amplification, is expanded by following degree：
（4）Result judgement：The period Ct value result of determination undergone according to fluorescence signal arrival given threshold：Work as the positive
The Ct values of reference substance be less than or equal to 35, or detection mixture for it is positive when, kit is effective；When the Ct values of positive reference substance are big
In 35, or when detection mixture is without Ct values, kit is invalid or need to repeat detection.
Wherein, step（4）In, qualitatively judged according to the Ct values of detection mixture, foundation is：During Ct ＞ 40, sample to be tested
Middle no HIV therapy medicine DDI and TDFF medicament-resistant mutation；During 35≤Ct≤40, sample to be tested is suspicious, need to examine again once, again inspection knot
Fruit such as Ct ＞ 40 or without amplification curve, then in sample to be tested without HIV therapy medicine DDI and TDFF medicament-resistant mutation, it is on the contrary then be
There is HIV therapy medicine DDI and TDFF medicament-resistant mutation in sample to be tested；During Ct ﹤ 35, have in sample to be tested HIV therapy medicine DDI and
TDFF medicament-resistant mutations produce.
The method of described detection treating AIDS medicine DDI and TDF resistant mutational site is RT-ARMS-Taqman
QPCR methods, this method are based on real-time quantitative PCR platform, and specific PCR amplification is carried out using ARMS primer pairs mutated target sequence,
Taqman probes carry out specific position detection to amplified production, and specific mutation is identified on the basis of Real-time PCR.
Beneficial effect：It is disclosed by the invention based on RT-ARMS-Taqman qPCR technology for detection HIV-1 drug resistances DI and
The method in TDF mutational sites, there are high specificity, high sensitivity, easy to operate, quick, low cost and other advantages.The ARMS of design
Primer is just for gene mutation sequence, and energy specific amplification mutagenesis template RNA, 3 ' in ARMS primers design to 5 ' end 1-2 positions
Two base mismatch, the specificity of ARMS primers can be increased, the Taqman probes that the present invention designs are for target gene justice
Mutational site design even, specifically with reference to mutagenesis template RNA, the ARMS primers of design just for gene specific mutant nucleotide sequence,
Energy specific amplification mutagenesis template RNA, so as to reach the enrichment to micro mutagenesis template, improve detection sensitivity, this hair
Bright disclosed method detection gene mutation sensitivity can reach 1%（That is the mutator RNA template numbers of target gene account for wild
The 1% of type RNA template numbers）, and the sensitivity of direct sequencing detection gene mutation is 10%（That is the mutator of target gene
RNA template numbers account for the 10% of wild type rna template number）, detection process is stopped pipe reaction, reduces the possibility of pollution, operation letter
Single quick, whole PCR courses of reaction only have 90 minutes, and as a result interpretation is clearly objective, need to be according to the data on PCR instruments only
Can complete paired samples gene type interpretation, while the present invention also have it is safe, whole system does not include venomous injurant
Matter, without the open pipe of PCR primer, to testing crew and environment all non-hazardous.
Brief description of the drawings
Fig. 1 is using the synthesis saltant type pol RT genes containing K65R point mutation as template, is carried out with different dilution ratios
PCR is expanded, and detects the RT-ARMS- of main resistant mutational site K65R, K70E and the Q151M saltant types of DDI and TDF two
Taqman qPCR testing result figures.
Fig. 2 is using the synthesis saltant type pol RT genes containing K70E point mutation as template, is carried out with different dilution ratios
PCR is expanded, and detects the RT-ARMS- of main resistant mutational site K65R, K70E and the Q151M saltant types of DDI and TDF two
Taqman qPCR testing result figures.
Fig. 3 is using the synthesis saltant type pol RT genes containing Q151M point mutation as template, is carried out with different dilution ratios
PCR is expanded, and detects the RT-ARMS- of main resistant mutational site K65R, K70E and the Q151M saltant types of DDI and TDF two
Taqman qPCR testing result figures.
Fig. 4 is the qPCR testing result figures for being expanded to obtain by template of positive reference substance.
Fig. 5 is the qPCR testing result figures for being expanded to obtain by template of negative controls.
Embodiment
Embodiment combination the drawings and specific embodiments are described further to such scheme.These embodiments are to be used to say
The bright present invention, the implementation condition used in embodiment can do further adjustment according to the explanation for producer of stealthily giving the glad eye, unreceipted
Implementation condition be usually condition in normal experiment.
The reagent and its source that the present invention uses：MLV and Taq enzyme are purchased from Dalian treasured biotech firm, and other reagents are not as made
Explanation is experiment conventional reagent.
Embodiment 1
For detecting the kit for the treatment of AIDS medicine DDI and TDF resistant mutational site, including：
（1）RT-PCR mixed liquors 875uL；
RT-PCR buffer solutions（20mM Tris-Hcl, 100mM NaCl, pH8.3）Final concentration 1 ×；
DNTPs final concentrations 3mM；
MgCl2Final concentration of 1.6mM；
3 pairs of primer pairs and 3 probes for being used to detect treating AIDS medicine DDI and TDF resistant mutational site：Each ARMS
The final concentration of the upstream and downstream primer of primer pair is 200nM；The Taqman and kit quality-control product primer of each ARMS primer pairs
To Taqman probe final concentrations be 300nM；
Specifically, included in the RT-PCR mixed liquors：Primer sequence SEQ ID No.1, SEQ ID No.2, SEQ
ID No.4, SEQ ID No.5, SEQ ID No.6 its each final concentration is respectively 0.2,0.4,0.2,0.2,0.2mmol/uL,
Taqman probe sequences are SEQ ID No.3, SEQ ID No.7, and final concentration is respectively 0.2,0.1mmol/uL；
SEQ ID No ：1
K65R:
F3: 5’-CTCCAGTATTTGCCATAAACTG-3’（Tm 56.5, length 22）
SEQ ID No ：2
R1: 5’-CTAGGTATGGTAAATGCAGTATAC-3’（Tm 52.1, length 24）
SEQ ID No ：3
A1: FAM/HEX-5’-CTGAAGTCTTCATCTAAGGGAACTGAA-3’-BHQ（Tm 62.4, length 27）
SEQ ID No ：4
F7: 5’-CATAAAGAAAAAAGACAGTACATG-3’（Tm 53, length 24）
SEQ ID No ：5
F15：5’-TATCAGTACAATGTGCTTCGTAT-3’ （Tm 54.3, length 23）
SEQ ID No ：6
R2: 5’-TACTGTCCATTTATCAGGATG-3’（Tm 52.7, length 21）
SEQ ID No ：7
A2: ROX-5’-CATAACCCATCCAAAGGAATGG-3’-BHQ（Tm 62.4, length 22）
Wherein, for detecting the primer pair and probe for the treatment of AIDS medicine DDI and TDF resistant mutational site, including inspection
Survey the 65th, 70 and 151 mutational site K65R, K70E and Q151M of the pol genes of HIV-1 viral RNAs ARMS primers
Pair and Taqman probes；
The sense primer and anti-sense primer of K65R ARMS primer pairs are respectively SEQ ID No.1 and SEQ ID No.2 institutes
The nucleotide sequence shown, Taqman probes are the nucleotide sequence shown in SEQ ID No.3, respectively with FAM and BHQ1 marks 5 '
End and 3 ' ends；
The sense primer and anti-sense primer of K70E ARMS primer pairs are respectively SEQ ID No.4 and SEQ ID No.2 institutes
The nucleotide sequence shown, Taqman probes are the nucleotide sequence shown in SEQ ID No.3, respectively with HEX and BHQ1 marks 5 '
End and 3 ' ends；
The sense primer and anti-sense primer of Q151M ARMS primer pairs are respectively SEQ ID No.5 and SEQ ID No.6 institutes
The nucleotide sequence shown, Taqman probes are the nucleotide sequence shown in SEQ ID No.7, respectively with ROX and BHQ1 marks 5 '
End and 3 ' ends.
（2）The uL of mixed enzyme solution 125；
M-MLV reverse transcriptase final concentration 2U/ μ L；
Taq DNA polymerase final concentration 0.05U/ μ L；
（3）The uL of positive quality control product 45；
（4）The uL of positive reference substance mixed liquor 45；
Template cDNA final concentrations 0.1~10ng/ μ L in positive reference substance mixed liquor；
The positive reference substance mixture is the mixture of three kinds of positive reference substances, including positive reference substance solution 1, is referred to logical
Cross artificial synthesized method K65R point mutation is introduced into HIV-1 pol RT genes and obtained, positive reference substance solution 2, refer to and pass through
K70E point mutation is introduced HIV-1 pol RT genes and obtained, positive reference substance solution 3 by artificial synthesized method, is referred to and is passed through people
Q151M point mutation is introduced HIV-1 pol RT genes and obtained by the method for work synthesis；Its preparation method, it is specific as follows：
HIV-1 pol RT gene cDNA sequences are as shown in SEQ ID No .8.
K65R point mutation is incorporated into by HIV-1 pol RT genes by artificial synthesized method, it is right to obtain the saltant type positive
According to product 1.The HIV-1 pol RT saltant types positive reference substance 1 of the point mutation containing K65R of synthesis is cloned into pGM-T carriers, structure
Recombinant plasmid is built up, then by recombinant plasmid transformed DH5 α Escherichia coli, after sequencing identification is correct, extraction recombinant plasmid dilution
Afterwards, positive reference substance 1 is made.
K70E point mutation is incorporated into by HIV-1 pol RT genes by artificial synthesized method, it is right to obtain the saltant type positive
According to product 2.The HIV-1 pol RT saltant types positive reference substance 2 of the point mutation containing K70E of synthesis is cloned into pGM-T carriers, structure
Recombinant plasmid is built up, then by recombinant plasmid transformed DH5 α Escherichia coli, after sequencing identification is correct, extraction recombinant plasmid dilution
Afterwards, positive reference substance 2 is made.
Q151M point mutation is incorporated into by HIV-1 pol RT genes by artificial synthesized method, obtains the saltant type positive
Reference substance 3.The HIV-1 pol RT saltant types positive reference substance 3 of the point mutation containing K70E of synthesis is cloned into pGM-T carriers,
Recombinant plasmid is built into, then by recombinant plasmid transformed DH5 α Escherichia coli, after sequencing identification is correct, extraction recombinant plasmid is dilute
After releasing, positive reference substance 3 is made.
Obtained positive reference substance 1, positive reference substance 2, positive reference substance 3 are respectively diluted to 1% concentration, are mixed into
Positive reference substance mixed liquor, it is standby.
（5）The uL of negative controls mixed liquor 45.
The negative controls mixed liquor is the healthy human body genomic DNA of extraction.
Treating AIDS medicine DDI and TDF the resistant mutational site RT-PCR detection kit pair of the embodiment 1 of embodiment 2
K65R detection sensitivity
Prepared by each μ L of sample RT-PCR reaction solutions 17.5, the μ L of mixed enzyme solution 2.5, by 1 positives reference substance of embodiment
The 1% of 1, the 0.01% of positive reference substance, the 0.0001% of positive reference substance, the 0.000001% of positive reference substance be diluted after make
Enter performing PCR reaction for masterplate, each diluted sample applied sample amount is 5uL, to determine inspection of the detection kit to K65R in the present invention
Survey sensitivity（The final concentration of each component is respectively 1 × RT-PCR buffer solutions, 3 mM dNTPs, 1.6 mM in 20 μ L systems
MgCl2,2U/ μ L M-MLV reverse transcriptases, 0.05 U/ μ L Taq DNA polymerases, 200nM primers, 300nM Taqman are visited
Pin）, PCR reaction conditions are：42 DEG C of min of reverse transcription 10, a circulation；95 DEG C of pre-degenerations 3 minutes, a circulation； 95℃
Denaturation 10 seconds, 55 DEG C are annealed and extended 40 seconds, 45 amplification cycles.As a result it is as shown in Figure 1.
As shown in Figure 1, detection method disclosed by the invention can reach to the detection sensitivity of K65R point mutation
0.000001% sudden change sample.
Treating AIDS medicine DDI and TDF the resistant mutational site RT-PCR detection kit pair of the embodiment 1 of embodiment 3
K70E detection sensitivity
Prepared by each μ L of sample RT-PCR reaction solutions 17.5, the μ L of mixed enzyme solution 2.5, by 1 positives control of embodiment
After the 1% of product 2, the 0.01% of positive reference substance, the 0.0001% of positive reference substance, the 0.000001% of positive reference substance is diluted
Enter performing PCR reaction as masterplate, each diluted sample applied sample amount is 5uL, to determine that detection kit is to K70E's in the present invention
Detection sensitivity（The final concentration of each component is respectively 1 × RT-PCR buffer solutions, 3 mM dNTPs, 1.6 in 20 μ L systems
MM MgCl2,2U/ μ L M-MLV reverse transcriptases, 0.05 U/ μ L Taq DNA polymerases, 200nM primers, 300nM Taqman
Probe）, PCR reaction conditions are：42 DEG C of min of reverse transcription 10, a circulation；95 DEG C of pre-degenerations 3 minutes, a circulation； 95
DEG C denaturation 10 seconds, 55 DEG C anneal simultaneously extend 40 seconds, 45 amplification cycles.As a result it is as shown in Figure 2.
As shown in Figure 2, detection method disclosed by the invention can reach to the detection sensitivity of K70E point mutation
0.000001% sudden change sample.
Treating AIDS medicine DDI and TDF the resistant mutational site RT-PCR detection kit pair of the embodiment 1 of embodiment 4
Q151M detection sensitivity
RT-PCR reaction solutions and mixed enzyme solution, by each μ L of sample RT-PCR reaction solutions 17.5, the μ L of mixed enzyme solution 2.5 match somebody with somebody
System, by the 1% of the positives reference substance 3 of embodiment 1, the 0.01% of positive reference substance, the 0.0001% of positive reference substance, positive control
The 0.000001% of product enters performing PCR as masterplate after being diluted and reacted, and each diluted sample applied sample amount is 5uL, to determine this
Detection sensitivity of the detection kit to Q151M in invention（The final concentration of each component is respectively 1 × RT- in 20 μ L systems
PCR buffer solutions, 3 mM dNTPs, 1.6 mM MgCl2,2U/ μ L M-MLV reverse transcriptases, 0.05 U/ μ L Taq DNA polymerizations
Enzyme, 200nM primers, 300nM Taqman probes）, PCR reaction conditions are：42 DEG C of min of reverse transcription 10, a circulation；95℃
Pre-degeneration 3 minutes, a circulation；95 DEG C are denatured 10 seconds, and 55 DEG C are annealed and extended 40 seconds, 45 amplification cycles.As a result such as
Shown in Fig. 3.
From the figure 3, it may be seen that detection method disclosed by the invention can reach to the detection sensitivity of Q151M point mutation
0.000001% sudden change sample.
Embodiment 5
Detected using the treating AIDS medicine DDI and TDF resistant mutational site RT-PCR detection kit of embodiment 1.
RT-PCR reaction solutions and mixed enzyme solution, by each μ L of sample RT-PCR reaction solutions 17.5, the μ L of mixed enzyme solution 2.5 match somebody with somebody
System, performing PCR amplification is entered to positive reference substance mixed liquor, and PCR reaction conditions are：42 DEG C of min of reverse transcription 10, a circulation；95
DEG C pre-degeneration 3 minutes, a circulation；95 DEG C are denatured 10 seconds, and 55 DEG C are annealed and extended 40 seconds, 45 amplification cycles.
As a result it is as shown in Figure 4.
As shown in Figure 4, the Ct values for the positive reference substance being equipped with detection kit are below 35.
Embodiment 6
Detected using the treating AIDS medicine DDI and TDF resistant mutational site RT-PCR detection kit of embodiment 1.
RT-PCR reaction solutions and mixed enzyme solution, by each μ L of sample RT-PCR reaction solutions 17.5, the μ L of mixed enzyme solution 2.5 match somebody with somebody
System, performing PCR amplification is entered to negative controls mixed liquor, and PCR reaction conditions are：42 DEG C of min of reverse transcription 10, a circulation；95
DEG C pre-degeneration 3 minutes, a circulation；95 DEG C are denatured 10 seconds, and 55 DEG C are annealed and extended 40 seconds, 45 amplification cycles.As a result
As shown in Figure 3.
As shown in Figure 5, detection method disclosed by the invention to normal person's genome without amplification.
Embodiment 7
The 100 HIV-1 patient for having produced drug-resistant phenotype RNA samples are taken as template, with reagent disclosed by the invention
Box and RT-ARMS-Taqman qPCR methods are detected, drawn used in detection to K65R, tri- point mutation of K70E, Q151M
Thing probe sequence is identical with the primer probe sequence used in embodiment 1.Simultaneously by this detection method compared with direct Sequencing.
Comprise the following steps：
（1）Total RNAs extraction：The RNA of sample to be tested is extracted, mentions template ribonucleic acid solution；
（2）Reactive component is prepared：Detection mixture, positive reference substance mixture and negative controls mixing are prepared respectively
Thing；Wherein, detect mixture and prepare a person-portion by PCR mixed liquors 17.5uL, mixed enzyme solution 2.5uL, template ribonucleic acid solution 5uL；Sun
Property reference substance mixture prepare a person-portion by PCR mixed liquors 17.5uL, mixed enzyme solution 2.5uL, positive reference substance mixed liquor 5uL；
Negative controls mixture prepares a people by PCR mixed liquors 17.5uL, mixed enzyme solution 2.5uL, negative controls mixed liquor 5uL
Part；
（3）Amplification：Respectively will detection mixture, positive quality control product mixture, positive reference substance mixture and negative control
Product mixture is put into quantitative real time PCR Instrument amplification, is expanded by following degree：
Result judgement：The period Ct value result of determination undergone according to fluorescence signal arrival given threshold：When positive matter
The Ct values of control product and positive reference substance be less than or equal to 35, or detection mixture for it is positive when, kit is effective；Work as positive quality control
The Ct values of product or positive reference substance be more than 35, or detection mixture without Ct values when, kit it is invalid or need to repeat detection.According to
The Ct values for detecting mixture qualitatively judge, and foundation is：It is resistance to without HIV therapy medicine DDI and TDFF in sample to be tested during Ct ＞ 40
Medicine is mutated；During 35≤Ct≤40, sample to be tested is suspicious, need to examine again once, examines result such as Ct ＞ 40 again or without amplification curve, then for
Without HIV therapy medicine DDI and TDFF medicament-resistant mutation in sample to be tested, it is on the contrary then for have in sample to be tested HIV therapy medicine DDI and
TDFF medicament-resistant mutations；During Ct≤35, there is the generation of HIV therapy medicine DDI and TDFF medicament-resistant mutation in sample to be tested.
Testing result is shown：Kit disclosed by the invention is detected in 100 parts of samples, detects that 12 samples have K65R
Mutational site, 5 samples have K70E mutational sites, and 25 samples have Q151M mutational sites；Actual PCR sequencing PCR detects 100 parts of samples
In this, detect that 10 samples there are K65R mutational sites, 3 samples there are K70E mutational sites, and 20 samples there are Q151M mutation
Site.
From Analysis of test results, kit and RT-ARMS-Taqman qPCR methods disclosed by the invention detect K65R,
The sensitivity of K70E, Q151M mutation is above direct sequencing.
SEQUENCE LISTING
<110>Applicant Jiangsu Fa Shida bio tech ltd
<120>For detect treating AIDS medicine DDI and TDF resistant mutational site primer pair and probe and its should
With
<130> SG20150302
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>The sense primer of K65R ARMS primer pairs
<400> 1
ctccagtatt tgccataaac tg 22
<210> 2
<211> 24
<212> DNA
<213>The anti-sense primer of the anti-sense primer of K65R ARMS primer pairs and K70E ARMS primer pairs
<400> 2
ctaggtatgg taaatgcagt atac 24
<210> 3
<211> 27
<212> DNA
<213>Taqman probes
<400> 3
ctgaagtctt catctaaggg aactgaa 27
<210> 4
<211> 24
<212> DNA
<213>The sense primer of K70E ARMS primer pairs
<400> 4
cataaagaaa aaagacagta catg 24
<210> 5
<211> 23
<212> DNA
<213>The sense primer of Q151M ARMS primer pairs
<400> 5
tatcagtaca atgtgcttcg tat 23
<210> 6
<211> 21
<212> DNA
<213>The anti-sense primer of Q151M ARMS primer pairs
<400> 6
tactgtccat ttatcaggat g 21
<210> 7
<211> 22
<212> DNA
<213>Taqman probes
<400> 7
cataacccat ccaaaggaat gg 22
<210> 8
<211> 1680
<212> DNA
<213>HIV-1 pol RT gene cDNA sequences
<400> 8
cccattagcc ctattgagac tgtaccagta aaattaaagc caggaatgga tggcccaaaa 60
gttaaacaat ggccattgac agaagaaaaa ataaaagcat tagtagaaat ttgtacagag 120
atggaaaagg aagggaaaat ttcaaaaatt gggcctgaaa atccatacaa tactccagta 180
tttgccataa agaaaaaaga cagtactaaa tggagaaaat tagtagattt cagagaactt 240
aataagagaa ctcaagactt ctgggaagtt caattaggaa taccacatcc cgcagggtta 300
aaaaagaaaa aatcagtaac agtactggat gtgggtgatg catatttttc agttccctta 360
gatgaagact tcaggaagta tactgcattt accataccta gtataaacaa tgagacacca 420
gggattagat atcagtacaa tgtgcttcca cagggatgga aaggatcacc agcaatattc 480
caaagtagca tgacaaaaat cttagagcct tttagaaaac aaaatccaga catagttatc 540
tatcaataca tggatgattt gtatgtagga tctgacttag aaatagggca gcatagaaca 600
aaaatagagg agctgagaca acatctgttg aggtggggac ttaccacacc agacaaaaaa 660
catcagaaag aacctccatt cctttggatg ggttatgaac tccatcctga taaatggaca 720
gtacagccta tagtgctgcc agaaaaagac agctggactg tcaatgacat acagaagtta 780
gtggggaaat tgaattgggc aagtcagatt tacccaggga ttaaagtaag gcaattatgt 840
aaactcctta gaggaaccaa agcactaaca gaagtaatac cactaacaga agaagcagag 900
ctagaactgg cagaaaacag agagattcta aaagaaccag tacatggagt gtattatgac 960
ccatcaaaag acttaatagc agaaatacag aagcaggggc aaggccaatg gacatatcaa 1020
atttatcaag agccatttaa aaatctgaaa acaggaaaat atgcaagaat gaggggtgcc 1080
cacactaatg atgtaaaaca attaacagag gcagtgcaaa aaataaccac agaaagcata 1140
gtaatatggg gaaagactcc taaatttaaa ctgcccatac aaaaggaaac atgggaaaca 1200
tggtggacag agtattggca agccacctgg attcctgagt gggagtttgt taatacccct 1260
cccttagtga aattatggta ccagttagag aaagaaccca tagtaggagc agaaaccttc 1320
tatgtagatg gggcagctaa cagggagact aaattaggaa aagcaggata tgttactaat 1380
agaggaagac aaaaagttgt caccctaact gacacaacaa atcagaagac tgagttacaa 1440
gcaatttatc tagctttgca ggattcggga ttagaagtaa acatagtaac agactcacaa 1500
tatgcattag gaatcattca agcacaacca gatcaaagtg aatcagagtt agtcaatcaa 1560
ataatagagc agttaataaa aaaggaaaag gtctatctgg catgggtacc agcacacaaa 1620
ggaattggag gaaatgaaca agtagataaa ttagtcagtg ctggaatcag gaaagtacta 1680
Claims (1)
1. the kit for detecting treating AIDS medicine DDI and TDF resistant mutational site, it is characterised in that：
RT-PCR mixed liquors 875uL；
The uL of mixed enzyme solution 125；
The uL of positive quality control product 45；
The uL of positive reference substance mixed liquor 45；
The uL of negative controls mixed liquor 45；
Wherein,
The RT-PCR mixed liquors include：
RT-PCR buffer solutions, Tris-Hcl containing 20mM, 100mM NaCl, pH8.3 final concentration 1 ×；
DNTPs final concentrations 3mM；
MgCl2Final concentration of 1.6mM；
3 pairs of upstream and downstream primers and 3 probes；The final concentration of the upstream and downstream primer of each ARMS primer pairs is 200nM；Each ARMS
The Taqman of primer pair and the Taqman probe final concentrations of kit quality-control product primer pair are 300nM；
3 pairs of upstream and downstream primers and 3 articles of probes include the 65th, 70 and 151 of the pol genes of detection HIV-1 viral RNAs
Position mutational site K65R, K70E and Q151M ARMS primer pairs and Taqman probes；
The sense primer and anti-sense primer of K65R ARMS primer pairs are respectively shown in SEQ ID No.1 and SEQ ID No.2
Nucleotide sequence, Taqman probes be SEQ ID No.3 shown in nucleotide sequence, respectively with FAM and BHQ1 marks 5 ' end and
3 ' ends；
The sense primer and anti-sense primer of K70E ARMS primer pairs are respectively shown in SEQ ID No.4 and SEQ ID No.2
Nucleotide sequence, Taqman probes be SEQ ID No.3 shown in nucleotide sequence, respectively with HEX and BHQ1 marks 5 ' end and
3 ' ends；
The sense primer and anti-sense primer of Q151M ARMS primer pairs are respectively shown in SEQ ID No.5 and SEQ ID No.6
Nucleotide sequence, Taqman probes be SEQ ID No.7 shown in nucleotide sequence, respectively with ROX and BHQ1 marks 5 ' end and
3 ' ends；
The mixed enzyme solution includes：
M-MLV reverse transcriptase final concentration 2U/ μ L；
Taq DNA polymerase final concentration 0.05U/ μ L；
Template cDNA final concentration 0.1~10ng/ μ L in the positive reference substance mixed liquor.
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| CN105624333A (en) * | 2016-03-04 | 2016-06-01 | 广西医科大学 | Primer for detecting main drug-resistant mutation sites of aids therapeutic nucleoside reverse transcriptase inhibitor and application thereof |
| CN108315407A (en) * | 2018-02-11 | 2018-07-24 | 广西医科大学 | The multi-PCR detection method and its primer sets for the treatment of AIDS pharmaceutical protein enzyme enzyme inhibitor medicament-resistant mutation |
| CN108330189A (en) * | 2018-02-11 | 2018-07-27 | 广西医科大学 | The primer for the treatment of AIDS drug NRTIs resistant mutational sites I54V and its application |
| CN110079635A (en) * | 2019-05-17 | 2019-08-02 | 江苏发士达生物科技有限公司 | For detecting the primer pair and probe and its application for the treatment of AIDS drug ABC resistant mutational site |
| CN110079636A (en) * | 2019-05-20 | 2019-08-02 | 江苏发士达生物科技有限公司 | For detecting the primer pair and probe and its application for the treatment of AIDS drug D4T and AZT resistant mutational site |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982473A (en) * | 2005-12-16 | 2007-06-20 | 天津生物芯片技术有限责任公司 | Gene chip for inspecting IIIV P-reverse transcriptase inhibiting resistance and its reagent kit |
| CN102312004A (en) * | 2011-09-23 | 2012-01-11 | 张晓光 | Detection method and kit for gene mutations and HIV-1 drug resistance mutation sites |
| CN103215378A (en) * | 2013-03-19 | 2013-07-24 | 浙江大学 | Method for detection of HIV-1 nucleoside inhibitor drug resistance mutation and kit |
| CN104911252A (en) * | 2015-01-25 | 2015-09-16 | 江苏发士达生物科技有限公司 | Primer pair and probe used for detecting AIDS treatment medicine 3TC and FTC drug-resistance mutation sites and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008062385A2 (en) * | 2006-11-24 | 2008-05-29 | Reginald Anthony Cilliers | Antiretroviral drug resistance testing |
| US20140106977A1 (en) * | 2012-06-04 | 2014-04-17 | Feng Gao | Simultaneous detection of multiple mutations |
| CN105452487B (en) * | 2013-05-31 | 2020-02-14 | 美国健康及人类服务部 | Real-time PCR point mutation assay for detecting HIV-1 resistance to antiviral drugs |
-
2015
- 2015-03-05 CN CN201510097715.8A patent/CN104911255B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1982473A (en) * | 2005-12-16 | 2007-06-20 | 天津生物芯片技术有限责任公司 | Gene chip for inspecting IIIV P-reverse transcriptase inhibiting resistance and its reagent kit |
| CN102312004A (en) * | 2011-09-23 | 2012-01-11 | 张晓光 | Detection method and kit for gene mutations and HIV-1 drug resistance mutation sites |
| CN103215378A (en) * | 2013-03-19 | 2013-07-24 | 浙江大学 | Method for detection of HIV-1 nucleoside inhibitor drug resistance mutation and kit |
| CN104911252A (en) * | 2015-01-25 | 2015-09-16 | 江苏发士达生物科技有限公司 | Primer pair and probe used for detecting AIDS treatment medicine 3TC and FTC drug-resistance mutation sites and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| systematic evaluation of allele-specific real-time PCR for the detection of minor HIV-1 variants with pol and env resistance mutations;Roger Paredes等;《Journal of Virological methods》;20071231;第146卷(第1-2期);摘要和表1 * |
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