CN108676916A - A kind of HIV usRNA and cell number dual quantitative real-time fluorescent PCR reagent case and its detection method - Google Patents
A kind of HIV usRNA and cell number dual quantitative real-time fluorescent PCR reagent case and its detection method Download PDFInfo
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Abstract
The invention discloses the dual real-time fluorescence PCR kits and its detection method of HIV usRNA and cell quantity in a kind of simultaneous quantitative peripheral blood lymphocytes, the dual real-time fluorescence quantification kit of HIV usRNA/ cell quantities in a kind of peripheral blood lymphocytes of the present invention, including restriction enzyme, restriction enzyme buffer, RT PCR mix, enzyme mixation, HIV usRNA primer sets, HIV usRNA probes, β actin primer sets, β actin probes and quality-control product.The present invention can realize while the HIV usRNA in human peripheral blood lymphocyte and cell quantity are used for quickly detecting, and clinically have the value of bigger.
Description
Technical field
The present invention relates to field of molecular biotechnology, and in particular to HIV in a kind of simultaneous quantitative peripheral blood lymphocytes
The dual quantitative real-time fluorescent PCR reagent case and its detection method of usRNA and cell quantity.
Background technology
The relevant HIV-1 RNA of cell are considered to have potentiality in the patient that infected by HIV carries out active antiretroviral therapy
Diagnosis marker.The viral RNA different more than 40 kinds is transcribed by the provirus being integrated in infected cell, and by most
The alternative splicing of first transcript, generates in the cell of inhibition of HIV infection.In the process of infection, there is the non-montage of about 9kb to turn
Originally (unspliced RNA, usRNA) and the incomplete montage transcript more than 4kb generate for record, and then encode the knot of translation virus
Structure albumen and auxilin Gag, Pol, Env, Vif, Vpr and Vpu.In addition to being used as translating Gag albumen and Gag-Pol polyproteins
Template outside, usRNA is used as inhereditary material geneome RNA in being also packaged into progeny virus.Although viral RNA is in blood plasma sometimes
Middle contents level is more stable, but is significantly increased always with the relevant usRNA contents levels of cells into close.Now with text
The level for showing HIV-1 usRNA in peripheral blood lymphocytes (PBMC) is offered, the virus levels of blood plasma can be replaced to detect, become
One strong antiretroviral treatment diagnostic flag.Biochemistry founder's Fischer et al. is proved in anti-reverse transcription disease
In malicious curer, the relevant HIV RNA of 90% or more PBMC are derived from the cell of new infection.More there is article to show cell phase
The HIV RNA of pass can be as the identification marking of challenge virus repository, as the monitor control index of Virus reproductivity, and can be with
Potential strategy is provided for the radical cure of auxiliary propulsion HIV.
There are many inventions being detected for HIV RNA now, as Chinese patent 200510023805.9 discloses
A kind of fluorescent quantitative detecting kit of human immune deficiency virus HIV-1 nucleic acid amplification, by using multi-primers to HIV-1
It is measured in real time, the kit of the disclosure of the invention has good detection sensitivity, can be in clinical serum or plasma specimen
HIV RNA carry out qualitative and quantitative detection, the effect of anti-HIV-1 medicines, can be also detected, but the invention only stops
In RNA levels, there is no be carried out at the same time quantitative detection to cell quantity.
For another example Chinese patent 201510494301.9 discloses a kind of quantitative detection examination of human immunodeficiency virus HIV-1
Agent box, the invention can directly will detection sample and sample process agent mixed processings, you can be used for subsequent detection, to be omitted from
It is more time saving, simple in sample the step of extraction purification nucleic acid molecules, but the invention is also to only reside within RNA levels pair
HIV-1 carries out quantitative detection, and there is no cell quantity and RNA are carried out quantitative detection together.
To HIV usRNA and cell number simultaneous quantitative, the work that inhibition of HIV replicates can be preferably judged with adjuvant clinical doctor
Jump degree, but lack HIV usRNA and the dual quantitative real-time fluorescence PCR of cell number in a kind of human peripheral blood lymphocyte at present
Kit and its method, this is because can also have the HIV DNA and various mRNA of cell expression in the cell, in cell
β-actin can express mRNA, to interference cell number is quantified.
The present invention can close the activity of primer by being chemically modified to primer, realize the thermal starting of PCR, when up to
When to certain temperature, the chemical group of modification falls off, primer activity recovery.Using this technology, may be implemented only to expand β-
The DNA of actin, without expanding its mRNA.HIV usRNA and cell number simultaneous quantitative are detected to realize, can assist facing
Bed doctor preferably judges the active degree that inhibition of HIV replicates.
Invention content
Regarding the issue above, the present invention provides HIV usRNA and cell in a kind of human peripheral blood lymphocyte
Quantity dual quantitative real-time fluorescent PCR reagent case and its detection method.
In order to achieve the above objectives, the present invention uses following technical proposal：
On the one hand, the present invention provides HIV usRNA in a kind of human peripheral blood lymphocyte and cell quantity are dual quantitative
Real-time fluorescent PCR reagent case, including restriction enzyme, restriction enzyme buffer, RT-PCR mix, enzyme mixation,
Primer sets, probe groups and quality-control product；
The enzyme mixation includes restriction enzyme, Taq enzyme and mmlv enzymes；
The primer sets are made of HIV usRNA primer sets and β-actin primer sets；
The probe groups are made of HIV usRNA probes and β-actin probes；
The HIV usRNA primer sets are by HIV usRNA-F1 sense primers, HIV usRNA-F2 sense primers, HIV
UsRNA-R1 downstream primers and HIV usRNA-R2 downstream primers composition；
β-actin the primer sets are made of β-actin-F sense primers and β-actin-R downstream primers；
The HIV usRNA-F1 sense primers have the nucleotide sequence of SEQ ID No.1；
The HIV usRNA-F2 sense primers have the nucleotide sequence of SEQ ID No.3；
The HIV usRNA-R1 downstream primers have the nucleotide sequence of SEQ ID No.2；
The HIV usRNA-R2 downstream primers have the nucleotide sequence of SEQ ID No.4；
β-actin-F the sense primers have the nucleotide sequence of SEQ ID No.6；
β-actin-R the downstream primers have the nucleotide sequence of SEQ ID No.7；
The HIV usRNA probes have the nucleotide sequence of SEQ ID No.5；
β-actin the probes have the nucleotide sequence of SEQ ID No.8.
Further, the RT-PCR mix include Tris-MOPS- buffered sodium citrates system, ammonium sulfate and chlorination
Potassium.
Further, the quality-control product includes positive quality control product and negative quality-control product, the positive quality control product be containing
The pseudovirus of HIV segments and the mixture of human cell, the feminine gender quality-control product is physiological saline.
On the other hand, the present invention provides HIV usRNA in a kind of human peripheral blood lymphocyte and cell quantity are dual fixed
Reaction condition is arranged in the detection method of the real-time fluorescent PCR reagent case of amount, wherein PCR amplification：Reverse transcription, pre-degeneration, 6~
The low rigorous cycle that low rigorous cycle, 40 temperature that the rigorous cycle of height, 8 temperature of 9 cycles reduce further decrease,
In be non-touchdown PCR；
Further, the PCR amplification system of the detection method includes the amplification system of 50 μ L：Including PCR Mix
Then 20 μ l of RNA to be detected are added in 27.5 μ L, 2.5 μ L of enzyme mixation, brief centrifugation；Also according to above-mentioned system, sun is set
Property control and negative control, positive quality control product or feminine gender 20 μ L of quality-control product is added and is expanded；
Further, fluorescent PCR instrument cycling condition be 45 DEG C 20~45 minutes, 94~96 DEG C 2 minutes；94~95 DEG C
15~30 seconds, 65~69 DEG C 30~75 seconds, 68~72 DEG C 30~40 seconds, 6~9 cycle；93~95 DEG C 15~20 seconds, 60
DEG C 30 seconds, 68~72 DEG C 30 seconds, 8 cycles；93~95 DEG C 15 seconds, 52~55 DEG C 30~60 seconds, 40 cycle；
Further, it is preferable to be：Fluorescent PCR instrument cycling condition be 45 DEG C 40 minutes, 94 DEG C 2 minutes；94 DEG C 15 seconds,
65 DEG C 30 seconds, 72 DEG C 30 seconds, 8 cycle；94 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, 8 cycle；94 DEG C 15 seconds,
53 DEG C 50 seconds, 40 cycle；Fluorescence is collected at 55 DEG C.
Compared with prior art, the invention has the advantages that：
(1) digestion that the present invention passes through specificity so that the HIV DNA in sample are digested digestion, and only expand it
In HIV usRNA, meanwhile, because primer has carried out chemical modification, when low temperature, does not have activity, in previous stage, that is, reverse transcription
Phase cell avoids the mRNA of amplifying cells oneself expression without amplification so that quantifying for cell is more accurate；
(2) present invention accurately can accurately survey the HIV activity in per unit cell (such as 1,000,000 cells)
It is fixed, so as to be quantified simultaneously to intracellular HIV usRNA and cell DNA in single tube, and avoid HIV DNA and β-
The interference of actin mRNA, to more accurately reflect the active degree of intracellular HIV.
(3) present invention can close the activity of primer, realize the thermal starting of PCR by being chemically modified to primer, when
When reaching certain temperature, the chemical group of modification falls off, primer activity recovery, using this technology, may be implemented only to expand β-
The DNA of actin detects HIV usRNA and cell number simultaneous quantitative to realize, can assist facing without expanding its mRNA
Bed doctor preferably judges the active degree that inhibition of HIV replicates.
Description of the drawings
Fig. 1 is the AFLP system of 4 Plays product of embodiment.
Fig. 2 is that the linear collection of illustrative plates in detection range is determined in embodiment 5.
Fig. 3 is the sensitivity test result to HIV usRNA in embodiment 6.
Fig. 4 is the specificity experiments in embodiment 7.
Fig. 5 is the result expanded using the β-actin primers modified without thermal starting in embodiment 8.
Fig. 6 is the result expanded using the β-actin primers modified through thermal starting under similarity condition in embodiment 8.
Fig. 7 is that restriction enzyme is not used in embodiment 9 to carry out digestion, the result directly expanded to nucleic acid.
After Fig. 8 is carries out digestion using restriction enzyme under similarity condition in embodiment 9 to nucleic acid, then expanded
As a result.
Specific implementation mode
Following embodiment only exists in illustrative purpose, is limited the scope of the invention without being intended to.
1 collection of specimens of embodiment and pretreatment
The present embodiment and kit are applicable in specimen types：Fresh peripheral blood lymphocytes sample；
Peripheral blood lymphocytes is acquired with lymphocyte separation medium under aseptic condition, is directly detected；
The extraction of RNA and β-actin DNA：
(1) sample cell is centrifuged, is washed with PBS；
(2) cell RNA extracts kit (centrifugal column type, article No. 74104, the trade name RNeasy of Qiagen are used
Mini), the extraction of method progress RNA to specifications, obtains the HIV RNA for being mixed with β-actin DNA and HIV DNA；
HIV usRNA and the dual quantitative real-time fluorescence PCR reagent of cell quantity in 2 human peripheral blood lymphocyte of embodiment
Box
It is dual quantitative real-time to present embodiments provide HIV usRNA and cell quantity in a kind of human peripheral blood lymphocyte
Fluorescent PCR kit, including restriction enzyme, restriction enzyme buffer, RT-PCR mix, enzyme mixation, primer sets,
Probe groups and quality-control product；
The enzyme mixation includes restriction enzyme 2U/uL, Taq enzyme 1U/ μ L and mmlv enzyme 8U/ μ L：
The primer sets are made of HIV usRNA primer sets and β-actin primer sets；
The probe groups are made of HIV usRNA probes and β-actin probes；
The HIV usRNA primer sets are by HIV usRNA-F1 sense primers, HIV usRNA-F2 sense primers, HIV
UsRNA-R1 downstream primers and HIV usRNA-R2 downstream primers composition；
β-actin the primer sets are made of β-actin-F sense primers and β-actin-R downstream primers；
The nucleotide sequence such as SEQ ID No.1 of the HIV usRNA-F1 sense primers；
The HIV usRNA-F2 sense primers have the nucleotide sequence of SEQ ID No.3；
The HIV usRNA-R1 downstream primers have the nucleotide sequence of SEQ ID No.2；
The HIV usRNA-R2 downstream primers have the nucleotide sequence of SEQ ID No.4；
β-actin-F the sense primers have the nucleotide sequence of SEQ ID No.6；
β-actin-R the downstream primers have the nucleotide sequence of SEQ ID No.7；
The HIV usRNA probes have the nucleotide sequence of SEQ ID No.5；
β-actin the probes have the nucleotide sequence of SEQ ID No.8；
The RT-PCR mix include Tris-MOPS- buffered sodium citrates system, ammonium sulfate and potassium chloride.
The Tris-MOPS- sodium citrate buffer solutions are that 3- (N- morpholines) propane sulfonic acid, Tris and trisodium citrate is mutually mixed
It closes；3- (N- morpholines) propane sulfonic acid：Tris：The molar ratio of trisodium citrate is 20: 10: 5.
HIV usRNA and the dual quantitative real-time fluorescence PCR reagent of cell quantity in 3 human peripheral blood lymphocyte of embodiment
The detection method of box
The present embodiment uses the real-time fluorescent PCR reagent case in embodiment 2, PCR amplification setting reaction item in the present embodiment
Part includes：Low rigorous cycle that reverse transcription, pre-degeneration, the rigorous cycle of height, 8 temperature of 8 cycles reduce, 40 temperature are into one
The low rigorous cycle reduced is walked, wherein being non-touchdown PCR；
The PCR amplification system of the detection method includes the amplification system of 50 μ L：Including PCR Mix27.5 μ L, enzyme are mixed
2.5 μ L of liquid are closed, then 20 μ l of RNA to be detected are added in brief centrifugation；Also according to above-mentioned system, positive control and the moon are set
Property control, positive quality control product or feminine gender 20 μ L of quality-control product is added and is expanded；
Wherein, fluorescent PCR instrument cycling condition is：45 DEG C 40 minutes, 94 DEG C 2 minutes；94 DEG C 15 seconds, 65 DEG C 30 seconds,
72 DEG C 30 seconds, 8 cycle；94 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, 8 cycle；94 DEG C 15 seconds, 53 DEG C 50 seconds,
40 cycles；Fluorescence is collected at 55 DEG C.
Embodiment 4：The expanding effect of kit standard product is tested
The present embodiment uses the detection method in real-time fluorescent PCR reagent case and embodiment 3 in embodiment 2, will contain
The pseudovirus of HIV segments and the mixture of human cell, are prepared into the plasmid standards for quantitation of various concentration as shown in table 1；
Table 1
Experimental result is as shown in Figure 1, be from left to right followed successively by 2.5 × 105、2.5×104、2.5x103、2.5×
102Copies/mL plasmid standards for quantitation, type used are the Quantistudio DX fluorescence quantitative PCR instruments of American AB I companies.
The linear test of 5 kit of embodiment
The present embodiment uses the detection method in real-time fluorescent PCR reagent case and embodiment 3 in embodiment 2, and Fig. 2 is
The present embodiment carries out data through availability inspection and multinomial regression analysis, and after precision test, determines in detection range
Linear collection of illustrative plates, as a result show linear coverage area be 25~2.5 × 105copies/106Cell, type used are American AB I
7500 fluorescence quantitative PCR instruments of company.
Sensitivity test of 6 kit of embodiment to HIV usRNA
Pseudovirus is diluted to 25copies/106Cell, total volume extract for 200ul, and it is anti-to be equivalent to each PCR
2 copy HIV-1 usRNA of middle detection are answered, using the detection in the real-time fluorescent PCR reagent case and embodiment 3 in embodiment 2
Method is detected HIV-1 usRNA, and testing result by being repeated several times as shown in figure 3, detected, minimum detection of the invention
Limit (2 copies of detection in each PCR reactions) recall rate is up to 96%.Type used is that 7500 fluorescence of American AB I companies are fixed
Measure PCR instrument.
It can be seen that detection 2 copies during there is the present invention fabulous sensitivity, detection limit to be reacted up to each PCR.
The specificity experiments of 7 kit of embodiment
The present embodiment is specificity experiments, with the detection in the real-time fluorescent PCR reagent case and embodiment 3 in embodiment 2
Method is detected HIV-1 ' negative ' specimens or healthy population sample, and ' negative ' specimens include for HBV, herpes simplex types 1 virus, 2
Herpes simplex virus type, varicella virus, Epstein-Barr virus, cytomegalovirus, 6 herpes simplex virus types, hepatitis A
Virus, Hepatitis C Virus, flavivirus, 1 type of human T-leukemia virus and 2 types, Coxsackie virus B 3 and large intestine
The nucleic acid of bacillus extraction is detected, and the results are shown in Figure 4, is shown as negative (100%), and type used is Bio Rad Laboratories
Cfx96 fluorescence quantitative PCR instruments；
As it can be seen that the real-time fluorescent PCR reagent case of the present invention has significant specificity.
Embodiment 8
It is from left to right followed successively by 2.5 × 10 using the β-actin primer pair various concentrations modified without thermal starting4、2.5×
103、2.5×102、2.5×101Cell/mL samples expand, and quantitative cell, the results are shown in Figure 5；
2.5 are from left to right followed successively by using the β-actin primer pairs various concentrations modified through thermal starting under similarity condition ×
104、2.5×103、2.5×102、2.5×101Cell/mL samples are expanded, and quantitative cell, the results are shown in Figure 6；
As a result show that the β-actin primers modified without thermal starting can amplify β-actin mRNA, can not to cell into
Row is quantitative.
Embodiment 9
Restriction enzyme is not used, digestion is carried out to nucleic acid, directly HIV-1 usRNA and β-actin DNA are expanded
Increase, HIV-1 usRNA are from left to right followed successively by 2.5 × 104、2.5×103、2.5×102、2.5×101Copies/mL, as a result
As shown in Figure 7；
It after carrying out digestion to nucleic acid using restriction enzyme under similarity condition, then is expanded, the results are shown in Figure 8；
The result shows that restriction enzyme is not used carries out digestion to nucleic acid, HIV DNA can be amplified, HIV is caused
The quantitative inaccuracy of usRNA.
Specific embodiment described herein is only an example for the spirit of the invention.Technology belonging to the present invention is led
The technical staff in domain can make various modifications or additions to the described embodiments or replace by a similar method
In generation, however, it does not deviate from the spirit of the invention or beyond the scope of the appended claims.
Sequence table
<110>Guangzhou Supbio Bio-Technology Co., Ltd.
<120>In a kind of peripheral blood lymphocytes HIV usRNA and the dual quantitative real-time fluorescent PCR reagent case of cell number and
Its detection method
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Claims (10)
1. HIV usRNA and the dual quantitative real-time fluorescent PCR reagent case of cell quantity in a kind of peripheral blood lymphocytes, special
Sign is：Including restriction enzyme, restriction enzyme buffer, RT-PCR mix, enzyme mixation, primer sets, probe groups
And quality-control product；
The enzyme mixation includes restriction enzyme, Taq enzyme and mmlv enzymes；
The RT-PCR mix, including Tris-MOPS- buffered sodium citrates system, ammonium sulfate and potassium chloride；
The primer sets are made of HIV usRNA primer sets and β-ctin primer sets；
The probe groups are made of HIV usRNA probes and β-actin probes；
The HIV usRNA primer sets are by HIV usRNA-F1 sense primers, HIV usRNA-F2 sense primers, HIV
UsRNA-R1 downstream primers and HIV usRNA-R2 downstream primers composition；
β-actin the primer sets are made of β-actin-F sense primers and β-actin-R downstream primers；
The HIV usRNA-F1 sense primers have the nucleotide sequence of SEQ ID No.1；
The HIV usRNA-F2 sense primers have the nucleotide sequence of SEQ ID No.3；
The HIV usRNA-R1 downstream primers have the nucleotide sequence of SEQ ID No.2；
The HIV usRNA-R2 downstream primers have the nucleotide sequence of SEQ ID No.4；
β-actin-F the sense primers have the nucleotide sequence of SEQ ID No.6；
β-actin-R the downstream primers have the nucleotide sequence of SEQ ID No.7；
The HIV usRNA probes have the nucleotide sequence of SEQ ID No.5；
β-actin the probes have the nucleotide sequence of SEQ ID No.8.
2. kit as described in claim 1, it is characterised in that：Can in a reaction tube simultaneously to HIV usRNA and
β-actin DNA are quantified.
3. kit as described in claim 1, it is characterised in that：By restriction enzyme, specificity is carried out to HIV DNA
Ground digests；
The restriction enzyme include Acc65I, AlwI, BamHI, BanI, BsaJI, BslI, BsmFI, BsrI, BstF5I,
BstYI, Cac8I, FokI, KpnI, MnlI, NlaIV, TaqII and TspRI.
4. the detection method of HIVusRNA and the dual quantitative real-time fluorescence PCR of cell quantity in a kind of peripheral blood lymphocytes,
It is characterized in that：The detection method is using the kit described in claim 1-3 any one to a kind of periphery hemolymph
HIV usRNA and cell quantity are detected in cell.
5. detection method as claimed in claim 4, it is characterised in that：Thermal starting modification is carried out to β-actin primer sets.
6. detection method as claimed in claim 4, it is characterised in that：Fluorescent PCR instrument cycling condition is 45 DEG C 20~45 points
Clock, 94~96 DEG C 2 minutes；94~95 DEG C 15~30 seconds, 65~69 DEG C 30~75 seconds, 68~72 DEG C 30~40 seconds, 6~9
A cycle；93~95 DEG C 15~20 seconds, 60 DEG C 30 seconds, 68~72 DEG C 30 seconds, 8 cycle；93~95 DEG C 15 seconds, 52~55
DEG C 30~60 seconds, 40 cycles.
7. detection method as claimed in claim 6, it is characterised in that：Fluorescent PCR instrument cycling condition be 45 DEG C 40 minutes, 94
DEG C 2 minutes；94 DEG C 15 seconds, 65 DEG C 30 seconds, 72 DEG C 30 seconds, 8 cycle；94 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds,
8 cycles；94 DEG C 15 seconds, 53 DEG C 50 seconds, 40 cycle；Fluorescence is collected at 55 DEG C.
8. real-time fluorescent PCR reagent case as described in claim 1, quality-control product therein includes positive quality control product and negative Quality Control
Product, the positive quality control product are the mixture of the pseudovirus containing HIV segments and human cell, and the feminine gender quality-control product is physiology
Brine.
9. real-time fluorescent PCR reagent case as described in claim 1, wherein the enzyme mixation includes restriction enzyme 2
~10U/ μ L, Taq enzyme 1~2U/ μ L, mmlv enzyme 8~16U/ μ L.
10. real-time fluorescent PCR reagent case as described in claim 1, wherein the enzyme mixation includes restriction enzyme
2U/ μ L, Taq enzyme 1U/ μ L, mmlv enzyme 8U/ μ L.
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| CN104846116A (en) * | 2015-04-14 | 2015-08-19 | 广州海力特生物科技有限公司 | PCR primer groups and probe used for detecting human immunodeficiency virus type I, kit containing PCR primer groups and probe, and detection method |
| CN107119148A (en) * | 2017-05-09 | 2017-09-01 | 广州海力特生物科技有限公司 | Detect the LTR DNA of HIV 12 PCR primer group, probe and its kit and detection method |
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| CN104846116A (en) * | 2015-04-14 | 2015-08-19 | 广州海力特生物科技有限公司 | PCR primer groups and probe used for detecting human immunodeficiency virus type I, kit containing PCR primer groups and probe, and detection method |
| CN107119148A (en) * | 2017-05-09 | 2017-09-01 | 广州海力特生物科技有限公司 | Detect the LTR DNA of HIV 12 PCR primer group, probe and its kit and detection method |
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| RUI S. SOARES等: "Strategies to quantify unspliced and multiply spliced mRNA expression in HIV-2 infection", 《JOURNAL OF VIROLOGICAL METHODS》 * |
| ZANCHETTA等: "Long-Term Decay of the HIV-1 Reservoir in HIV-1–Infected Children Treated with Highly Active Antiretroviral Therapy", 《THE JOURNAL OF INFECTIOUS DISEASES》 * |
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