CN106967849A - The kit of HIVusRNA real time fluorescent quantitatives and its RT PCR detection methods in a kind of PBLC - Google Patents
The kit of HIVusRNA real time fluorescent quantitatives and its RT PCR detection methods in a kind of PBLC Download PDFInfo
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- CN106967849A CN106967849A CN201710322949.7A CN201710322949A CN106967849A CN 106967849 A CN106967849 A CN 106967849A CN 201710322949 A CN201710322949 A CN 201710322949A CN 106967849 A CN106967849 A CN 106967849A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
- C12Q1/702—Specific hybridization probes for retroviruses
- C12Q1/703—Viruses associated with AIDS
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses the kit and its RT PCR detection methods of HIV usRNA real time fluorescent quantitatives in a kind of PBLC, the kit of HIV usRNA real time fluorescent quantitatives in a kind of PBLC of the present invention, including RT PCR mix, enzyme mixation, primer sets, probe and quality-control product, the primer sets are made up of nucleotide sequence SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, and the probe is nucleotide sequence SEQ ID No.5.The present invention can realize that the HIV usRNA in human peripheral blood lymphocyte are used for quickly detecting, and detection sensitivity is good, using extremely convenient.
Description
Technical field
It is real more particularly, to HIV usRNA in a kind of PBLC the present invention relates to field of molecular biotechnology
When fluorescent quantitation kit and its RT-PCR detection method.
Background technology
The concentration (plasma viral load) of cell free virus in blood plasma, by the copy number of the viral RNA of every milliliter of blood plasma
Represent, it can be reliably quantitative by PCR method, and always as the traditional biological mark of HIV-1 virus replications.It is degeneration-resistant
The main target of retroviral therapy is, in order to reduce plasma viral load, the virus in blood plasma to be reduced by suppressing virus replication
Amount, and the standard that clinically most of sensitive detection methods can be detected is made it below, and treatment is kept this standard.
In most of HIV-1 the infecteds for receiving and adhering to ART, this target has been realized in, therefore in blood plasma
Virus load than relatively low, even below in detectable limit.Even if having passed through best treatment, in most of patient's body
Still remain low-level viral copies.Because low-level plasma viral detects challenging, therefore low-level is not known
Viremia virusemia whether can produce reaction to long-term treatment, the appearance of new diagnosis marker is also just very necessary.And cell
Related HIV-1RNA is considered to have the diagnosis marker of potentiality in the patient that infected by HIV carries out active antiretroviral therapy.
The viral RNA different more than 40 kinds is transcribed by the provirus being integrated in infected cell, and by initially turning
This alternative splicing is recorded, is produced in the cell that inhibition of HIV infects.In the process of infection, there is about 9kb non-montage transcript
(unspliced RNA) and the incomplete montage transcript generation more than 4kb, and then encode the viral structural proteins of translation and auxiliary
Help Protein G ag, Pol, Env, Vif, Vpr and Vpu.In addition to being used as translating the template of Gag albumen and Gag-Pol polyproteins,
UsRNA is also packaged into progeny virus as inhereditary material geneome RNA.Although viral RNA content water in blood plasma sometimes
More stable but related to cells into close usRNA contents levels are put down to significantly increase always.Proved now with document,
HIV-1usRNA level in PBLC (PBMC), can replace the virus levels of blood plasma to detect, strong as one
Strong ART diagnostic flag.The proof such as Fischer is in ART person, and more than 90%
HIV RNA related PBMC are derived from the cell of new infeetioa.More there is article to show, the related HIV RNA of cell can conduct
The identification marking of challenge virus repository, is used as the monitor control index of Virus reproductivity, it is possible to be auxiliary propulsion HIV radical cure
Potential strategy is provided.
The Patient cells of most of ART related RNA and DNA detection means is real-time PCR.
In recent years, PCR (RT-qPCR) was applied to quantifying for cell correlation HIV-1RNA.This method has very high specificity,
But after ART, in order to detect micro nucleic acid virus, the sensitiveness of detection means also needs to add
By force.Our primed probes special by designing can precisely detect the side of micro viral copies there is provided a kind of herein
Method.
There is provided a stable buffer system is most important to course of reaction for PCR.Tris-HCl buffer systems are
Most classical PCR working reaction systems, ensure the function such as pH stability in PCR work.But Tris-HCl buffer solutions are a kind of
Dual-polarized ion buffer solution, pKa is 8.3 (20 DEG C), and △ pKa are 0.021/ DEG C, and relative temperature is more sensitive, with the liter of temperature
High pH reductions.Under typical thermal cycle conditions, when elongating temperature is at 72 DEG C, real pH value is between 6.8-7.8, in pH
Less than less than 7.5, buffer capacity extreme difference.For many thermal starting enzymes, pH changes cause enzymatic activity to decline or even inactivate, influence
PCR or RT-PCR detection sensitivity and precision.
At present, a kind of kit of HIV usRNA real time fluorescent quantitatives in PBLC of quick detection is lacked
And its RT-PCR detection method.
The content of the invention
Regarding the issue above, the present invention provides HIV in a kind of PBLC of quick detection
The kit and its RT-PCR detection method of usRNA real time fluorescent quantitatives.
To reach above-mentioned purpose, present invention employs following technical proposal：In a kind of PBLC of the present invention
The kit of HIV usRNA real time fluorescent quantitatives, including RT-PCR mix, enzyme mixation, primer sets, probe and quality-control product, institute
Primer sets are stated to be made up of nucleotide sequence SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4,
The HIV usRNA-F1 sense primers have SEQ ID No.1 nucleotide sequence；
The HIV usRNA-F2 sense primers have SEQ ID No.3 nucleotide sequence；
The HIV usRNA-R1 anti-sense primers have SEQ ID No.2 nucleotide sequence；
The HIV usRNA-R2 anti-sense primers have SEQ ID No.4 nucleotide sequence；
The probe has SEQ ID No.5 nucleotide sequence.
Further, the quality-control product includes positive quality control product and negative quality-control product, and the positive quality control product is pseudovirus,
The negative quality-control product is physiological saline.
Further, described RT-PCR mix include Tris-MOPS- buffered sodium citrates system, ammonium sulfate and chlorination
Potassium.
Further, the Tris-MOPS- sodium citrate buffer solutions are 3- (N- morpholines) propane sulfonic acid, Tris and lemon
Sour trisodium, which is mutually mixed, to be formed；3- (N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 20-100：10-
50：1-5.
The PCR amplification bodies of the kit of HIV usRNA real time fluorescent quantitatives in PBLC of the present invention
System, the real-time fluorescence quantitative PCR augmentation detection system includes 50 μ L amplification system：50 μ L systems, include PCR Mix27.5
Then μ L, the μ L of enzyme mixation 2.5, brief centrifugation add the μ l of RNA 20 to be detected；Set positive right also according to above-mentioned system
According to and negative control, add positive quality control product or the feminine gender μ L of quality-control product 20 and expanded.
The detection method of the kit of HIV usRNA real time fluorescent quantitatives in a kind of PBLC of the present invention,
The PCR amplifications set reaction condition to include：High rigorous circulation (the non-landing circulated including reverse transcription, pre-degeneration, 6~9
PCR), low rigorous circulation (the non-landing that the low rigorous circulation (non-touchdown PCR) of 8 temperature reductions, 40 temperature are further reduced
PCR)。
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 circulation；93~95 DEG C 15~20 seconds, 60 DEG C 30 seconds,
68~72 DEG C 30 seconds, 8 circulation；93~95 DEG C 15 seconds, 52~55 DEG C 30~60 seconds, 40 circulation.
Further, fluorescent PCR instrument cycling condition be 45 DEG C 40 minutes, 94 DEG C 2 minutes；94 DEG C 15 seconds, 65 DEG C of 30 seconds, 72
DEG C 30 seconds, 8 circulations；94 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, 8 circulation；94 DEG C 15 seconds, 53 DEG C 50 seconds, 40 circulation；
Fluorescence is collected at 55 DEG C.
The kit and its detection method of HIV usRNA real time fluorescent quantitatives in PBLC of the present invention
Application in detection PBLC.
Beneficial effect：The present invention can realize that the HIV usRNA in human peripheral blood lymphocyte are used for quickly detecting, detection
Sensitivity is good, using extremely convenient.It is more suitable for the Tris-MOPS- sodium citrate buffer solutions of RNA amplification due to introducing so that
The sensitivity and specificity of detection are significantly strengthened, thus avoid conventional RT-PCR method specificity it is not high, with
And quantitative shortcoming can not be carried out to low concentration masterplate.
Compared with prior art, the invention has the advantages that：
(1) HIV usRNA are considered as valuable biomarker.Research at present on HIV usRNA is a lot, but
Commercialized usRNA detection reagents and method are not seen in clinical practice.
(2) MOPS is a kind of chemical substance for being usually used in preparing neutral buffer systems, and Chinese name is also known as 3- (N- morpholines) third
Sulfonic acid, solution state between pKa 72 (25 DEG C) buffering range 6.5-7.9, is widely used in two-dimensional gel electrophoresis medium into acidity
The electrolyte system composition of electric focusing, it may also be used for Northern hybridize, as RNA separation and transferring film when buffering
Liquid.
Brief description of the drawings
Fig. 1 is from left to right followed successively by 2.5 × 10 for the present invention5、2.5×104、2.5×103、2.5×102、2.5×101Copy
The collection of illustrative plates of the amplification of shellfish/mL standard items；
Fig. 2 carries out data through availability inspection and multinomial regression analysis for the present invention, and after precision test, it is determined that
Linear collection of illustrative plates in detection range；
Fig. 3 is that the sensitivity that HIV usRNA real-time fluorescence quantitative PCR detection methods of the present invention are detected to HIV usRNA is real
Test the collection of illustrative plates of result；
Such as figures of the Fig. 4 for the specificity experiments detected to HIV-1 ' negative ' specimens or healthy population sample of the present invention
Spectrum.
Embodiment
Following examples only exist in illustrative purpose, without being intended to limit the scope of the present invention.
Embodiment 1
The kit of HIV usRNA real time fluorescent quantitatives in a kind of PBLC of the present invention, including RT-PCR
Mix, enzyme mixation, primer sets, probe and quality-control product, the primer sets are by nucleotide sequence SEQ ID No.1, SEQ ID
No.2, SEQ ID No.3, SEQ ID No.4 are constituted,
The HIV usRNA-F1 sense primers have SEQ ID No.1 nucleotide sequence；
The HIV usRNA-F2 sense primers have SEQ ID No.3 nucleotide sequence；
The HIV usRNA-R1 anti-sense primers have SEQ ID No.2 nucleotide sequence；
The HIV usRNA-R2 anti-sense primers have SEQ ID No.4 nucleotide sequence；
The probe has SEQ ID No.5 nucleotide sequence.
The quality-control product includes positive quality control product and negative quality-control product, and the positive quality control product is pseudovirus, the feminine gender
Quality-control product is physiological saline.
Described 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
Conjunction is formed；3- (N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 20：40：1.
The PCR amplification bodies of the kit of HIV usRNA real time fluorescent quantitatives in PBLC of the present invention
System, the real-time fluorescence quantitative PCR augmentation detection system includes 50 μ L amplification system：50 μ L systems, include PCR Mix27.5
Then μ L, the μ L of enzyme mixation 2.5, brief centrifugation add the μ l of RNA 20 to be detected；Set positive right also according to above-mentioned system
According to and negative control, add positive quality control product or the feminine gender μ L of quality-control product 20 and expanded.
The detection method of the kit of HIV usRNA real time fluorescent quantitatives in a kind of PBLC of the present invention,
The PCR amplifications set reaction condition to include：High rigorous circulation (the non-landing circulated including reverse transcription, pre-degeneration, 6~9
PCR), low rigorous circulation (the non-landing that the low rigorous circulation (non-touchdown PCR) of 8 temperature reductions, 40 temperature are further reduced
PCR)。
Fluorescent PCR instrument cycling condition be 45 DEG C 20 minutes, 94~96 DEG C 2 minutes；94~95 DEG C 15 seconds, 65~69 DEG C 75
Second, 68~72 DEG C 30 seconds, 6~9 circulation；93~95 DEG C 20 seconds, 60 DEG C 30 seconds, 68~72 DEG C 30 seconds, 8 circulation；93~95
DEG C 15 seconds, 52~55 DEG C 30 seconds, 40 circulations.
The kit and its detection method of HIV usRNA real time fluorescent quantitatives in PBLC of the present invention
Application in detection PBLC.
Reality using the present invention using pseudovirus as plasmid standards for quantitation HIV usRNA real-time fluorescence quantitative PCR detection methods
Test, Fig. 1 is from left to right followed successively by 2.5 × 10 for the present invention5、2.5×104、2.5×103、2.5×102、2.5×101Copy/mL
Standard items amplification collection of illustrative plates；Type：ABI7500.
Examined as shown in Fig. 2 Fig. 2 carries out data for the present invention through availability inspection and multinomial regression analysis, and through precision
After testing, it is determined that the linear collection of illustrative plates in detection range；This reagent result is linear；Final result shows that linear coverage is 25
~2.5 × 105copies/ml；Type：ABI7500.
Fig. 3 is tested for minimum detectability, and pseudovirus is diluted into 25copies/ml, and cumulative volume is that 200ul is extracted
(equivalent to 2 copy HIV-1usRNA of detection in each PCR reactions), are examined using above detection method to HIV-1usRNA
Survey；Testing result by being repeated several times as shown in figure 3, detected, minimum detectability of the invention (detects 2 in each PCR reactions
Copy) recall rate is up to 96%.Type：ABI 7500.
As shown in figure 4, being the specificity experiments of the present invention, HIV-1 ' negative ' specimens or healthy population sample are detected,
' negative ' specimens include for HBV, herpes simplex types 1 virus, herpes simplex types 2 virus, varicella virus, Epstein-Barr virus, carefully
Born of the same parents' hugeization virus, 6 herpes simplex virus types, hepatitis A virus, HCV, flavivirus, the white blood of human T cells
The nucleic acid that viral 1 type of disease and 2 types, Coxsackie virus B 3 and Escherichia coli are extracted is detected, is as a result shown as negative
(100%).Result above proves that the inventive method has good specificity.Type：Bole CFX96.
Embodiment 2
The difference of embodiment 2 and embodiment 1 is：
The Tris-MOPS- sodium citrate buffer solutions are that 3- (N- morpholines) propane sulfonic acid, Tris and trisodium citrate is mutually mixed
Conjunction is formed；3- (N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 100：10：4.
Fluorescent PCR instrument cycling condition be 45 DEG C 35 minutes, 94~96 DEG C 2 minutes；94~95 DEG C 30 seconds, 65~69 DEG C 30
Second, 68~72 DEG C 40 seconds, 6~9 circulation；93~95 DEG C 15 seconds, 60 DEG C 30 seconds, 68~72 DEG C 30 seconds, 8 circulation；93~95
DEG C 15 seconds, 52~55 DEG C 60 seconds, 40 circulations.
Embodiment 3
The difference of embodiment 3 and embodiment 1 is：
The Tris-MOPS- sodium citrate buffer solutions are that 3- (N- morpholines) propane sulfonic acid, Tris and trisodium citrate is mutually mixed
Conjunction is formed；3- (N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 80：50：5.
Fluorescent PCR instrument cycling condition be 45 DEG C 45 minutes, 94~96 DEG C 2 minutes；94~95 DEG C 20 seconds, 65~69 DEG C 65
Second, 68~72 DEG C 35 seconds, 6~9 circulation；93~95 DEG C 18 seconds, 60 DEG C 30 seconds, 68~72 DEG C 30 seconds, 8 circulation；93~95
DEG C 15 seconds, 52~55 DEG C 50 seconds, 40 circulations.
Experiment 1
The design of specific primer and probe：
According to the HIV genomic nucleic acid sequences retrieved from Genbank, selection LTR regions, designed for detection HIV
UsRNA primer sets and probe, its sequence is as follows：
Primer sets：
HIV usRNA-F1：5’-TACACACCAGGGCCAGGGAT-3’(SEQ ID NO.1)
HIV usRNA-R1：5’-ACAGGGTGTAACAAGCTGGTGT-3’(SEQ ID NO.2)
HIV usRNA-F2：5’-ACCTTTGGATGGTGCTACAA-3’(SEQ ID NO.3)
HIV usRNA-R2：5’-TGTTCTCTCCTTTGTTGGCT-3’(SEQ ID NO.4)
Fluorescence probe：
HIV usRNA–P：5’-Fam-
ACCAGTTGAGCCAGAGAAGT-3 ' BHQ1 (SEQ ID NO.5),
Expanding fragment length is 76bp；
Collection of specimens and pretreatment：
This method and kit are applicable specimen types：Fresh PBLC sample.
PBLC is gathered with lymphocyte separation medium under aseptic condition, is directly detected, or Liquid nitrogen storage is to be checked.
RNA extraction：
(1) sample cell is centrifuged, is washed with PBS；
(2) using Qiagen cell RNA extracts kit (centrifugation column type), according to saying
Bright book method carries out RNA extraction.And carry out the digestion of DNA on film；
Fluorescent quantitative PCR：
Each test reaction system is formulated as follows, 50 μ L systems, includes PCR Mix (setting experimental group and control group) 27.5
μ l, brief centrifugation, then add the μ l of RNA 20 to be detected；Also according to above-mentioned system, positive and negative control is set.
Described RT-PCR mix include Tris-MOPS- buffered sodium citrates system, ammonium sulfate and potassium chloride.It is described
Tris-MOPS- sodium citrate buffer solutions are that 3- (N- morpholines) propane sulfonic acid, Tris and trisodium citrate is mutually mixed and formed；It is described
3- (N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 20-100：10-50：1-5.
The PCR Mix (experimental group) be table 1 shown in,
Table 1
Each reaction tube is put into the reactive tank of quantitative PCR instruments, sets the title and fluorophor species of each detection (to set
The reporter group for putting HIV usRNA is FAM, quenching group selection none), set cycling condition：
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 of 30 seconds, 8
Individual circulation；94 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, 8 circulation；94 DEG C 15 seconds, 53 DEG C 50 seconds, 40 circulation；Received at 55 DEG C
Collect fluorescence.
Specific embodiment described herein is only to spirit explanation for example of the invention.Technology neck belonging to of the invention
The technical staff in domain can be made various modifications or supplement to described specific embodiment or be replaced using similar mode
Generation, but without departing from the spiritual of the present invention or surmount scope defined in appended claims.
Claims (9)
1. the kit of HIV usRNA real time fluorescent quantitatives in a kind of PBLC, it is characterised in that：Including RT-PCR
Mix, enzyme mixation, primer sets, probe and quality-control product, the primer sets are by nucleotide sequence SEQ ID No.1, SEQ ID
No.2, SEQ ID No.3, SEQ ID No.4 are constituted,
The HIV usRNA-F1 sense primers have SEQ ID No.1 nucleotide sequence；
The HIV usRNA-F2 sense primers have SEQ ID No.3 nucleotide sequence；
The HIV usRNA-R1 anti-sense primers have SEQ ID No.2 nucleotide sequence；
The HIV usRNA-R2 anti-sense primers have SEQ ID No.4 nucleotide sequence；
The probe has SEQ ID No.5 nucleotide sequence.
2. the kit of HIV usRNA real time fluorescent quantitatives, its feature in PBLC according to claim 1
It is：The quality-control product includes positive quality control product and negative quality-control product, and the positive quality control product is pseudovirus, the negative Quality Control
Product are physiological saline.
3. the kit of HIV usRNA real time fluorescent quantitatives, its feature in PBLC according to claim 1
It is：Described RT-PCR mix include Tris-MOPS- buffered sodium citrates system, ammonium sulfate and potassium chloride.
4. the kit of HIV usRNA real time fluorescent quantitatives, its feature in PBLC according to claim 3
It is：The Tris-MOPS- sodium citrate buffer solutions are that 3- (N- morpholines) propane sulfonic acid, Tris and trisodium citrate is mutually mixed
Form；3- (N- morpholines) propane sulfonic acid：Tris：The mol ratio of trisodium citrate is 20-100：10-50：1-5.
5. the PCR amplification bodies of the kit of HIV usRNA real time fluorescent quantitatives in the PBLC described in claim 1
System, it is characterised in that：The real-time fluorescence quantitative PCR augmentation detection system includes 50 μ L amplification system：50 μ L systems, comprising
Then PCR Mix27.5 μ L, the μ L of enzyme mixation 2.5, brief centrifugation add the μ l of RNA 20 to be detected；Also according to above-mentioned body
System sets positive control and negative control, adds positive quality control product or the feminine gender μ L of quality-control product 20 are expanded.
6. the detection method of the kit of HIV usRNA real time fluorescent quantitatives in a kind of PBLC, it is characterised in that：
The PCR amplifications set reaction condition to include：Including reverse transcription, pre-degeneration, 6~9 circulation it is high it is rigorous circulate, 8 temperature
The low rigorous circulation that the low rigorous circulation of reduction, 40 temperature are further reduced.
7. the detection of the kit of HIV usRNA real time fluorescent quantitatives in PBLC according to claim 6
Method, it is characterised in that：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 circulation；93~95 DEG C 15~20 seconds, 60 DEG C 30 seconds,
68~72 DEG C 30 seconds, 8 circulation；93~95 DEG C 15 seconds, 52~55 DEG C 30~60 seconds, 40 circulation.
8. the detection of the kit of HIV usRNA real time fluorescent quantitatives in PBLC according to claim 7
Method, 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 of 30 seconds, 72
DEG C 30 seconds, 8 circulations；94 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, 8 circulation；94 DEG C 15 seconds, 53 DEG C 50 seconds, 40 circulation；
Fluorescence is collected at 55 DEG C.
9. in the PBLC described in any one of claim 1 to 8 kit of HIV usRNA real time fluorescent quantitatives and
Application of its detection method in detection PBLC.
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