CN106967849B - The kit and its RT-PCR detection method of HIVusRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes - Google Patents
The kit and its RT-PCR detection method of HIVusRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes Download PDFInfo
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Abstract
The invention discloses the kits and its RT-PCR detection method of HIV usRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes, the kit of HIV usRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes of the invention, including RT-PCR mix, enzyme mixation, primer sets, probe and quality-control product, the primer sets are made 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 be realized the HIV usRNA in human peripheral blood lymphocyte and be used for quickly detecting, and detection sensitivity is good, and application is extremely convenient.
Description
Technical field
The present invention relates to field of molecular biotechnology, real more particularly, to HIV usRNA in a kind of peripheral blood lymphocytes
When fluorescent quantitation kit and its RT-PCR detection method.
Background technique
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
It indicates, can be reliably quantitative by PCR method, and always as the traditional biological marker of HIV-1 virus replication.It is degeneration-resistant
The main target of retroviral therapy is to reduce the virus in blood plasma by suppressing virus replication to reduce plasma viral load
Amount, and the standard that the detection method for making it below clinically most of sensitivities can detect, and treatment is made to keep this standard.In
In most of HIV-1 the infecteds for receiving and adhering to antiretroviral treatment, this target is had been realized in, therefore in blood plasma
Virus load it is relatively low, even lower than in detectable limit.Even if having passed through best treatment, most of patient's body
Still remain low-level viral copies.Since low-level plasma viral detects challenging, unclear low-level
Viremia virusemia whether can to long-term treatment generate reaction, the appearance of new diagnosis marker also just it is very necessary.And cell
Relevant 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 turning to initial
The alternative splicing for recording this generates in the cell of inhibition of HIV infection.In the process of infection, there is the non-montage transcript of about 9kb
(unspliced RNA) and incomplete montage transcript greater than 4kb generate, and then encode the structural proteins of translation virus and auxiliary
Help Protein G ag, Pol, Env, Vif, Vpr and Vpu.Other than being used as translating the template of Gag albumen and Gag-Pol polyprotein,
UsRNA be also packaged into progeny virus as inhereditary material geneome RNA.Although viral RNA content water in blood plasma sometimes
Put down more stable, but usRNA contents level relevant to cells into close increases significantly always.It is proved now with document,
The level of HIV-1usRNA in peripheral blood lymphocytes (PBMC) can replace the virus levels detection of blood plasma, become one strong
Strong antiretroviral treatment diagnostic flag.The proof such as Fischer is in antiretroviral treatment person, and 90% or more
The relevant HIV RNA of PBMC is derived from the cell of new infection.More there is article to show that the relevant HIV RNA of cell can be used as
The identification marking of challenge virus repository as the monitor control index of Virus reproductivity, and can be the radical cure of auxiliary propulsion HIV
Potential strategy is provided.
The detection means of the Patient cells of most of antiretroviral treatment relevant RNA and DNA is real-time PCR.In
In recent years, PCR (RT-qPCR) had been applied to quantifying for cell correlation HIV-1RNA.This method has very high special
Property, but after antiretroviral treatment, in order to detect micro nucleic acid virus, the sensibility of detection means also needs
Reinforce.We, which pass through, herein designs special primed probe, and micro viral copies can precisely be detected by providing one kind
Method.
For PCR, it is most important to reaction process to provide a stable buffer system.Tris-HCl buffer system
It is most classic PCR working reaction system, ensures the functions such as stability of pH in PCR work.But Tris-HCl buffer is one
The dual-polarized ion buffer of kind, pKa are 8.3 (20 DEG C), and △ pKa is 0.021/ DEG C, and relative temperature is more sensitive, with temperature
Raising pH reduce.Under typical thermal cycle conditions, when elongating temperature is at 72 DEG C, real pH value between 6.8-7.8,
In pH less than 7.5 hereinafter, buffer capacity is very poor.For many thermal starting enzymes, pH variation causes enzymatic activity decline even to inactivate,
Influence the detection sensitivity and precision of PCR or RT-PCR.
Currently, lacking the kit of HIV usRNA real time fluorescent quantitative in the peripheral blood lymphocytes that one kind quickly detects
And its RT-PCR detection method.
Summary of the invention
Regarding the issue above, the present invention provides HIV in a kind of peripheral blood lymphocytes quickly detected
The kit and its RT-PCR detection method of usRNA real time fluorescent quantitative.
In order to achieve the above objectives, present invention employs following technical proposals: in a kind of peripheral blood lymphocytes of the invention
The kit of HIV usRNA real time fluorescent quantitative, including RT-PCR mix, enzyme mixation, primer sets, probe and quality-control product, institute
Primer sets are stated to be made of nucleotide sequence SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4,
The HIV usRNA-F1 upstream primer has the nucleotide sequence of SEQ ID No.1；
The HIV usRNA-F2 upstream primer has the nucleotide sequence of SEQ ID No.3；
The HIV usRNA-R1 downstream primer has the nucleotide sequence of SEQ ID No.2；
The HIV usRNA-R2 downstream primer has the nucleotide sequence of SEQ ID No.4；
The probe has the nucleotide sequence of SEQ ID No.5.
Further, 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.
Further, the RT-PCR mix includes Tris-MOPS- buffered sodium citrate system, ammonium sulfate and chlorination
Potassium.
Further, the Tris-MOPS- sodium citrate buffer solution is 3- (N- morpholine) propane sulfonic acid, Tris and lemon
Sour trisodium is mutually mixed；3- (N- morpholine) propane sulfonic acid: Tris: the molar ratio of trisodium citrate is 20-100:10-
50:1-5.
The PCR amplification body of the kit of HIV usRNA real time fluorescent quantitative in peripheral blood lymphocytes of the present invention
System, the real-time fluorescence quantitative PCR augmentation detection system includes the amplification system of 50 μ L: 50 μ L systems, includes PCR
Then 20 μ l of RNA to be detected is added in Mix27.5 μ L, 2.5 μ L of enzyme mixation, brief centrifugation；It is set also according to above-mentioned system
Positive control and negative control are set, positive quality control product is added or feminine gender 20 μ L of quality-control product is expanded.
The detection method of the kit of HIV usRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes of the invention,
The PCR amplification setting reaction condition includes: rigorous circulation (the non-landing of height including reverse transcription, initial denaturation, 6~9 circulations
PCR), low rigorous circulation (the non-landing that the low rigorous circulation (non-touchdown PCR) of 8 temperature reductions, 40 temperature further decrease
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
Second, 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 30 seconds,
72 DEG C 30 seconds, 8 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 are followed
Ring；Fluorescence is collected at 55 DEG C.
The kit and its detection method of HIV usRNA real time fluorescent quantitative in peripheral blood lymphocytes of the present invention
Application in detection peripheral blood lymphocytes.
It is used for quickly detecting, examines the utility model has the advantages that the present invention can be realized the HIV usRNA in human peripheral blood lymphocyte
Survey sensitivity is good, and application is extremely convenient.It is more suitable for the Tris-MOPS- sodium citrate buffer solution of RNA amplification due to introducing, makes
The sensitivity and specificity that must be detected significantly are enhanced, not high so as to avoid conventional RT-PCR method specificity,
And quantitative disadvantage can not be carried out to low concentration template.
Compared with prior art, the present invention has the advantage that
(1) HIV usRNA is considered as valuable biomarker.There are many research at present about HIV usRNA, but
Commercialized usRNA detection reagent and method are not seen in clinical application.
(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- morpholine) third
Between pKa 72 (25 DEG C) buffering range 6.5-7.9, it is medium to be widely used in two-dimensional gel electrophoresis at acidity for sulfonic acid, solution state
The electrolyte system ingredient of electric focusing, it may also be used for Northern hybridization, buffering when separation and transferring film as RNA
Liquid.
Detailed description of the invention
Fig. 1 is that the present invention is from left to right followed successively by 2.5 × 105、2.5×104、2.5×103、2.5×102、 2.5×101
The map of the amplification of copy/mL standard items；
Fig. 2 is that the present invention carries out data through availability inspection and multinomial regression analysis, and after precision test, determines
Linear map in detection range；
Fig. 3 is that the sensitivity that HIV usRNA real-time fluorescence quantitative PCR detection method of the present invention detects HIV usRNA is real
Test the map of result；
Such as the figure that Fig. 4 is the specificity experiments of the invention detected to HIV-1 ' negative ' specimens or healthy population sample
Spectrum.
Specific embodiment
Following embodiment only exists in illustrative purpose, limits the scope of the invention without being intended to.
Embodiment 1
The kit of HIV usRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes of the 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 composition,
The HIV usRNA-F1 upstream primer has the nucleotide sequence of SEQ ID No.1；
The HIV usRNA-F2 upstream primer has the nucleotide sequence of SEQ ID No.3；
The HIV usRNA-R1 downstream primer has the nucleotide sequence of SEQ ID No.2；
The HIV usRNA-R2 downstream primer has the nucleotide sequence of SEQ ID No.4；
The probe has the nucleotide sequence of SEQ ID No.5.
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.
The RT-PCR mix includes Tris-MOPS- buffered sodium citrate system, ammonium sulfate and potassium chloride.
The Tris-MOPS- sodium citrate buffer solution is that 3- (N- morpholine) propane sulfonic acid, Tris and trisodium citrate are mutual
It mixes；3- (N- morpholine) propane sulfonic acid: Tris: the molar ratio of trisodium citrate is 20:40:1.
The PCR amplification body of the kit of HIV usRNA real time fluorescent quantitative in peripheral blood lymphocytes of the present invention
System, the real-time fluorescence quantitative PCR augmentation detection system includes the amplification system of 50 μ L: 50 μ L systems, includes PCR
Then 20 μ l of RNA to be detected is added in Mix27.5 μ L, 2.5 μ L of enzyme mixation, brief centrifugation；It is set also according to above-mentioned system
Positive control and negative control are set, positive quality control product is added or feminine gender 20 μ L of quality-control product is expanded.
The detection method of the kit of HIV usRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes of the invention,
The PCR amplification setting reaction condition includes: rigorous circulation (the non-landing of height including reverse transcription, initial denaturation, 6~9 circulations
PCR), low rigorous circulation (the non-landing that the low rigorous circulation (non-touchdown PCR) of 8 temperature reductions, 40 temperature further decrease
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 quantitative in peripheral blood lymphocytes of the present invention
Application in detection peripheral blood lymphocytes.
Using the present invention using pseudovirus as the reality of plasmid standards for quantitation HIV usRNA real-time fluorescence quantitative PCR detection method
It tests, Fig. 1 is that the present invention is from left to right followed successively by 2.5 × 105、2.5×104、 2.5×103、2.5×102、2.5×101Copy/
The map of the amplification of the standard items of mL；Type: ABI7500.
As shown in Fig. 2, Fig. 2 is that present invention progress data are examined through availability inspection and multinomial regression analysis, and through precision
After testing, the linear map in detection range is determined；This reagent result is linear；Final result shows that linear coverage area is 25
~2.5 × 105copies/ml；Type: ABI7500.
Fig. 3 is minimum detectability experiment, pseudovirus is diluted to 25copies/ml, total volume extracts for 200ul
(be equivalent in each PCR reaction and detect 2 copy HIV-1usRNA), examines HIV-1usRNA using the above detection method
It surveys；Testing result is as shown in figure 3, by the way that detection is repeated several times, minimum detectability of the invention (detects 2 in each PCR reaction
A copy) recall rate is up to 96%.Type: ABI 7500.
As shown in figure 4, being specificity experiments of the 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, Hepatitis C Virus, flavivirus, mankind's T cell are white
The nucleic acid that viral 1 type of blood disease and 2 types, Coxsackie virus B 3 and Escherichia coli extract is detected, and is as the result is shown feminine gender
(100%).Result above proves that the method for the present invention has specificity well.Type: Bole CFX96.
Embodiment 2
Embodiment 2 the difference from embodiment 1 is that:
The Tris-MOPS- sodium citrate buffer solution is that 3- (N- morpholine) propane sulfonic acid, Tris and trisodium citrate are mutual
It mixes；3- (N- morpholine) propane sulfonic acid: Tris: the molar 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
Embodiment 3 the difference from embodiment 1 is that:
The Tris-MOPS- sodium citrate buffer solution is that 3- (N- morpholine) propane sulfonic acid, Tris and trisodium citrate are mutual
It mixes；3- (N- morpholine) propane sulfonic acid: Tris: the molar 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.
Test 1
The design of specific primer and probe:
According to the HIV genomic nucleic acid sequence retrieved from Genbank, the region LTR is selected, designed for detection HIV
The primer sets and probe of usRNA, sequence are 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 in specimen types: fresh peripheral blood lymphocytes sample.
Peripheral blood lymphocytes is acquired with lymphocyte separation medium under aseptic condition, directly detection or Liquid nitrogen storage are to be checked.
The extraction of RNA:
(1) it is centrifuged sample cell, is washed with PBS；
(2) the cell RNA extracts kit (centrifugal column type) for using Qiagen, according to saying
Bright book method carries out the extraction of RNA.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
Then 20 μ l of RNA to be detected is added in μ l, brief centrifugation；Also according to above-mentioned system, positive and negative control is set.
The RT-PCR mix includes Tris-MOPS- buffered sodium citrate system, ammonium sulfate and potassium chloride.It is described
Tris-MOPS- sodium citrate buffer solution is that 3- (N- morpholine) propane sulfonic acid, Tris and trisodium citrate is mutually mixed；It is described
3- (N- morpholine) propane sulfonic acid: Tris: the molar 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, the title and fluorophor type that each detection is arranged (are set
The reporter group for setting HIV usRNA is FAM, and quenching group selects 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 30 seconds, 8
A 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；At 55 DEG C
Collect fluorescence.
Specific embodiment described herein is only an example for the spirit of the invention.The neck of technology belonging to the present invention
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.
Claims (6)
1. the kit of HIV usRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes, 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 composition,
The HIV usRNA-F1 upstream primer has the nucleotide sequence of SEQ ID No.1；
The HIV usRNA-F2 upstream primer has the nucleotide sequence of SEQ ID No.3；
The HIV usRNA-R1 downstream primer has the nucleotide sequence of SEQ ID No.2；
The HIV usRNA-R2 downstream primer has the nucleotide sequence of SEQ ID No.4；
The probe has the nucleotide sequence of SEQ ID No.5；
The RT-PCR mix includes Tris-MOPS- buffered sodium citrate system, ammonium sulfate and potassium chloride；
The Tris-MOPS- sodium citrate buffer solution for 3- (N- morpholine) propane sulfonic acid, Tris and trisodium citrate is mutually mixed and
At；3- (N- morpholine) propane sulfonic acid: Tris: the molar ratio of trisodium citrate is 20-100:10-50:1-5.
2. the kit of HIV usRNA real time fluorescent quantitative, feature in peripheral blood lymphocytes according to claim 1
Be: 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 are physiological saline.
3. the kit of HIV usRNA real time fluorescent quantitative, feature in peripheral blood lymphocytes according to claim 2
Be: the PCR amplification detection architecture of the kit includes the amplification system of 50 μ L: 50 μ L systems, includes PCR Mix27.5 μ
L, then 20 μ L of RNA to be detected is added in 2.5 μ L of enzyme mixation, brief centrifugation；It is positive right also according to the setting of above-mentioned system
According to and negative control, positive quality control product or feminine gender 20 μ L of quality-control product is added and is expanded.
4. the kit of HIV usRNA real time fluorescent quantitative is used for non-disease in peripheral blood lymphocytes as described in claim 1
The detection method of sick diagnostic purpose, it is characterised in that: in the detection method PCR amplification setting reaction condition include: reverse transcription,
Low rigorous circulation, 40 temperature that initial denaturation, the rigorous circulation of height, 8 temperature of 6~9 circulations reduce further decrease low
Rigorous circulation；The rigorous circulation of height of 6~9 circulations for 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；Low rigorously the following of 8 temperature reduction
Ring be 93~95 DEG C 15~20 seconds, 60 DEG C 30 seconds, 68~72 DEG C 30 seconds, 8 circulation；40 temperature further decrease low
Rigorous circulation for 93~95 DEG C 15 seconds, 52~55 DEG C 30~60 seconds, 40 circulations.
5. detection method according to claim 4, 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 circulation；94 DEG C 15 seconds, 60 DEG C 30 seconds, 72 DEG C 30 seconds, 8 are followed
Ring；94 DEG C 15 seconds, 53 DEG C 50 seconds, 40 circulation；Fluorescence is collected at 55 DEG C.
6. the described in any item kits of claim 1-3 and detection method described in claim 4 or 5 diagnose mesh in non-disease
Detection peripheral blood lymphocytes in application.
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| CN201710322949.7A CN106967849B (en) | 2017-05-09 | 2017-05-09 | The kit and its RT-PCR detection method of HIVusRNA real time fluorescent quantitative in a kind of peripheral blood lymphocytes |
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Non-Patent Citations (3)
| Title |
|---|
| Hong F et al..Novel Assays for measurement of total cell-associated HIV-1 DNA.《J.Clin Microbiol》.2016,902-911. * |
| Maria J Buzon et al..HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects.《Nature Medicine》.2010,第16卷(第4期),online methods. * |
| Pasternak et al..Cell-associated HIV RNA: a dynamic biomarker of viral persistence.《Retrovirology》.2013,41. * |
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