CN111534640A - Reagent and method for qualitative detection of HIV - Google Patents

Reagent and method for qualitative detection of HIV Download PDF

Info

Publication number
CN111534640A
CN111534640A CN202010396986.4A CN202010396986A CN111534640A CN 111534640 A CN111534640 A CN 111534640A CN 202010396986 A CN202010396986 A CN 202010396986A CN 111534640 A CN111534640 A CN 111534640A
Authority
CN
China
Prior art keywords
seq
primer
eclipse
hiv
actb
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202010396986.4A
Other languages
Chinese (zh)
Other versions
CN111534640B (en
Inventor
姚均
蒋岩
王爱玲
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Maternal And Child Health Center Chinese Center For Disease Control And Prevention
NATIONAL CENTER FOR AIDS/STD CONTROL AND PREVENTION CHINESE CENTER FOR DISEASE CONTROL AND PREVENTION
Original Assignee
Maternal And Child Health Center Chinese Center For Disease Control And Prevention
NATIONAL CENTER FOR AIDS/STD CONTROL AND PREVENTION CHINESE CENTER FOR DISEASE CONTROL AND PREVENTION
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Maternal And Child Health Center Chinese Center For Disease Control And Prevention, NATIONAL CENTER FOR AIDS/STD CONTROL AND PREVENTION CHINESE CENTER FOR DISEASE CONTROL AND PREVENTION filed Critical Maternal And Child Health Center Chinese Center For Disease Control And Prevention
Priority to CN202010396986.4A priority Critical patent/CN111534640B/en
Publication of CN111534640A publication Critical patent/CN111534640A/en
Application granted granted Critical
Publication of CN111534640B publication Critical patent/CN111534640B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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
    • 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]
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • AIDS & HIV (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a group of primer pairs for detecting HIV by multiplex fluorescence PCR amplification reaction, a kit containing the primer pairs and used for detecting HIV by multiplex fluorescence PCR amplification reaction, and a method for detecting HIV by multiplex fluorescence PCR amplification reaction by using the primer pairs or the kit. By the invention, the detection lower limit of 10cps can be realized, and the method is more accurate compared with the existing detection method.

Description

Reagent and method for qualitative detection of HIV
Technical Field
The invention relates to the field of human AIDS virus detection, in particular to a group of primer pairs for multiplex fluorescence PCR amplification reaction, a kit comprising the primer pairs and a method for detecting AIDS virus by using the kit.
Background
Human Immunodeficiency Virus (HIV), also known as AIDS Virus, is a double-stranded RNA retrovirus that selectively invades the Human immune system after entering the Human body4+T lymphocytes and macrophages, in the cell, synthesize cDNA via a reverse transcription mechanism, form a pre-integration complex and integrate into the host cell's genomic DNA, which is called DNA proviral DNA. HIV-1RNA is present in two forms of the same substance as DNA provirus, depending on the life history of the HIV virus. However, DNA provirus is more stable than RNA because of its integration into the human genome, and because DNA provirus and HIV-1RNA gene have the same base sequence and can be detected simultaneously in PCR reaction. In medical studies, the HIV virus free in plasma is commonly referred to as HIV-RNA, while the HIV virus from peripheral blood immune cells is referred to as DNA provirus. The HIV genome is about 9.2-9.7 kb in length, and contains three structural genes including GAG, POL and Env and at least six regulatory genes.
The laboratory detection technique of HIV is extremely important for the diagnosis, treatment and epidemic monitoring of AIDS. There are several methods for detecting HIV, generally classified into immunological and virological tests. The immunological detection is to indirectly diagnose HIV infection by detecting HIV antibody, and comprises an enzyme-linked immunosorbent assay, a chemiluminescence assay, a rapid assay and the like according to the principle. As a main means for diagnosing AIDS, immunological detection is currently widely used in various fields such as clinic, emergency treatment, blood screening and the like. However, antibodies are usually detected 3-8 weeks after infection, so the immunological method is an indirect and delayed detection method because the target of detection is antibodies produced in the human body and not the pathogen itself. Compared with an immunological detection method, virological detection of AIDS has the characteristics of strong specificity and high sensitivity, thereby being beneficial to realizing early diagnosis by shortening the detection window period and avoiding missing detection, and being the main direction for continuously developing HIV diagnosis methods and diagnosis reagents. More importantly, the HIV-positive pregnant women give babies who, because of their maternal IGG antibodies, last up to 18 months, and during this time, the detection of antibodies does not allow the determination of the baby's infection status, but only virological detection methods.
At present, most of the kits for HIV gene detection are products for detecting HIV-1RNA in plasma, for example, Roche "human immunodeficiency virus (type 1) nucleic acid detection kit
Figure BDA0002487936640000021
AmpliPrep/
Figure BDA0002487936640000022
HIV-1Qualitative Test, version2.0) detection kit "is HIV nucleic acid Qualitative detection, but the target substance detected by the kit is still HIV-1RNA, the strain detected by the kit is mainly European and American popular B subtype, RNA is reverse transcribed to generate complementary DNA (cDNA) in the detection process, and false positive occurs when CT value is large (more than or equal to 33) after amplification. For another example, the method of the prior art, CN106119413A, using Long Terminal Repeats (LTR) sequence and GAG structural protein gene as primers, amplified both regions simultaneously during detection, had improved sensitivity and specificity but still had the problem of false positives.
Therefore, a detection technology with improved detection sensitivity and specificity for qualitative detection of HIV nucleic acid is needed, which can not only solve the problem of false positive of the existing kit, but also be suitable for early diagnosis of infection of HIV-positive puerpera-born exposed infants, differential diagnosis of HIV antibody-uncertain people, detection of HIV antibody window, supplementary test of antibody detection, inspection and quarantine, epidemic prevention and control and other fields.
Disclosure of Invention
The invention aims to provide a scheme for detecting the HIV with higher sensitivity and specificity. After a large number of reference sequences are analyzed and compared, long terminal repetitive sequence LTR, GAG and POL structural genes of HIV-1 are selected as target genes and amplified through multiple fluorescence PCR, so that the nucleic acid qualitative detection of the HIV is realized, and the invention is completed.
Accordingly, in a first aspect, the present invention provides a set of primer pairs for detecting hiv by multiplex fluorescent PCR amplification reactions, wherein the primer pairs comprise:
LTR upstream primer: 5'-AAGGCTACTTCCCTGATT-3' (SEQ ID NO: 1);
LTR downstream primer: 5'-TTGGCTTCTTCTAACTTCTC-3' (SEQ ID NO: 2);
GAG upstream primer: 5'-CAGAATGGGATAGAGTGC-3' (SEQ ID NO: 4);
GAG downstream primer: 5'-TACTGGGATAGGTGGATT-3' (SEQ ID NO: 5);
POL upstream primer: 5'-TAAGACAGCAGTACAAATGGCAG-3' (SEQ ID NO: 7);
POL downstream primer: 5'-GCTGTCCCTGTAATAAACCCG-3' (SEQ ID NO: 8).
In a second aspect, the present invention provides a kit for detecting HIV by multiplex fluorescent PCR amplification reactions, wherein the kit comprises the primer pair of the first aspect.
In a third aspect, the present invention provides a method for detecting HIV by a multiplex fluorescent PCR amplification reaction, said method for non-diagnostic purposes, comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) taking a sample DNA to be detected as a template, and carrying out multiple fluorescence PCR amplification reaction by using the primer pair of the first aspect or the kit of the second aspect;
(3) judging the result of the multiplex fluorescence PCR amplification reaction:
if the Ct values of two or three of the three genes to be amplified are less than 35 and the amplification curve is S-shaped, then the existence of the AIDS virus is judged,
if none of the three amplified genes presents an amplification curve or a Ct value, determining that the HIV does not exist,
and if the Ct value of only one of the three amplified genes is less than 35 and the amplification curve is in an S type, determining that whether the AIDS virus exists or not.
In a fourth aspect, the present invention provides the use of the primer pair of the first aspect in the preparation of a detection agent for detecting an aids virus nucleic acid.
The invention has the advantages that:
the invention has extremely high sensitivity, and even if only AIDS virus as low as 10cps exists in the sample, the invention can also carry out correct detection on the AIDS virus; the detection time is short, and the detection result can be obtained only in 3 hours; the invention has broad-spectrum specificity and can effectively and correctly detect various subtypes of HIV-1; the invention has extremely high detection accuracy, no selectivity to the type of the sample and the detection result is also accurate even for the dry blood spot sample.
Drawings
The technical solutions and benefits of the present invention will become apparent to those skilled in the art upon a reading of the following detailed description and a review of the associated drawings.
FIG. 1 shows the results of sensitivity (FAM) of LTR gene detection by multiplex fluorescence PCR according to an embodiment of the present invention.
FIG. 2 shows the sensitivity results (HEX) of the multiplex fluorescence PCR detection of GAG genes according to one embodiment of the present invention.
FIG. 3 is a graph showing the results of sensitivity of the detection of POL gene by multiplex fluorescence PCR according to an embodiment of the present invention (CY 5).
FIG. 4 is a graph showing typical results of multiplex fluorescence PCR detection of HIV-positive infected persons according to one embodiment of the present invention.
FIG. 5 is a graph showing typical results of multiplex fluorescence PCR detection of HIV-negative infected persons according to one embodiment of the present invention.
Detailed Description
The present invention is described in detail below. It is to be understood that the following description is intended to illustrate the present invention by way of example only and is not intended to limit the scope of the invention, which is defined by the appended claims. Also, it is understood by those skilled in the art that modifications may be made to the technical aspects of the present invention without departing from the spirit and gist of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
As described above, there is a need in the art for a detection technique with improved detection sensitivity and specificity for the qualitative detection of HIV nucleic acid. The present inventors have conducted an analysis and comparison of a large number of reference sequences, and then have selected long terminal repeat LTR, GAG and POL structural genes of HIV-1 as target genes, and amplified them by multiplex fluorescence PCR, thereby achieving a qualitative nucleic acid detection for HIV, and thus completed the present invention.
Accordingly, in a first aspect, the present invention provides a set of primer pairs for detecting hiv by multiplex fluorescent PCR amplification reactions, wherein the primer pairs comprise:
LTR upstream primer: 5'-AAGGCTACTTCCCTGATT-3' (SEQ ID NO: 1);
LTR downstream primer: 5'-TTGGCTTCTTCTAACTTCTC-3' (SEQ ID NO: 2);
GAG upstream primer: 5'-CAGAATGGGATAGAGTGC-3' (SEQ ID NO: 4);
GAG downstream primer: 5'-TACTGGGATAGGTGGATT-3' (SEQ ID NO: 5);
POL upstream primer: 5'-TAAGACAGCAGTACAAATGGCAG-3' (SEQ ID NO: 7);
POL downstream primer: 5'-GCTGTCCCTGTAATAAACCCG-3' (SEQ ID NO: 8).
The primer sequence of the present invention can be synthesized by a method known to those skilled in the art, and is not particularly limited.
In one embodiment, the primer pair may further include a primer pair for β -actin (β -action, abbreviated as ACTB):
ACTB upstream primer: 5'-ATTTGGACCTGCGAGCG-3' (SEQ ID NO:10)
ACTB downstream primer: 5'-AGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 11).
Beta-actin is a protein which is constantly expressed in a human body, and the gene thereof serves as an internal reference in the present invention to detect whether a reaction system is established.
In a second aspect, the present invention provides a kit for detecting HIV by multiplex fluorescent PCR amplification reaction, wherein the kit comprises the primer pair of the first aspect.
As known to those skilled in the art, for the fluorescent PCR amplification reaction, at least two methods can be adopted to realize the fluorescent detection, the first method is to add a fluorescent dye into the amplification product after the PCR amplification reaction is finished for dyeing, so as to perform corresponding judgment; the second method is to add a fluorescent probe to the PCR amplification system, thereby performing detection in real time.
Thus, in a specific embodiment, the kit further comprises the following fluorescent probes:
LTR fluorescent probe: 5'-FAM-CAAGCTAGTACCAGTT-ECLIPSE-3' (SEQ ID NO: 3);
GAG fluorescent probe: 5'-HEX-GAGTGCATCCAGTGCAT-ECLIPSE-3' (SEQ ID NO: 6);
POL fluorescent probe: 5'-CY5-ATTACAGGGACAGCAGAA-ECLIPSE-3' (SEQ ID NO: 9).
In the above fluorescent probe, FAM, HEX and CY5 are fluorescent reporter groups, and ECLIPSE is a fluorescent quencher group.
As noted above, in certain embodiments, β -actin is also used as an internal control. In such embodiments, the kit may further comprise:
ACTB fluorescent probe:
5'-TEXASRED-CTGACCTGAAGGCTCTGC-ECLIPSE-3'(SEQ ID NO:12)。
similarly, TEXASRED is a fluorescent reporter group.
It is understood that aids DNA positive and negative quality controls may also be included in embodiments of the invention to further ensure the accuracy of the detection system. The source or others of the positive and negative quality control materials are not particularly limited, and may be, for example, positive and negative quality control materials prepared using 8E5 cells.
In a third aspect, the present invention provides a method for detecting HIV by a multiplex fluorescent PCR amplification reaction, said method for non-diagnostic purposes, comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) performing a multiplex fluorescence PCR amplification reaction by using a sample DNA to be detected as a template and using the primer pair of the first aspect of the invention or the kit of the second aspect of the invention;
(3) judging the result of the multiplex fluorescence PCR amplification reaction:
if the Ct values of two or three of the three genes to be amplified are less than 35 and the amplification curve is S-shaped, then the existence of the AIDS virus is judged,
if none of the three amplified genes presents an amplification curve or a Ct value, determining that the HIV does not exist,
and if the Ct value of only one of the three amplified genes is less than 35 and the amplification curve is in an S type, determining that whether the AIDS virus exists or not.
The sample to be tested can be a whole blood sample or a dry blood spot sample, such as a sample from a healthy person, a known infected person of HIV, a suspected infected person of HIV, or a baby born by a parturient infected with HIV. The extraction of the DNA from the test sample can be carried out by conventional methods known to those skilled in the art, for example, by QIAGEN, Germany
Figure BDA0002487936640000071
DNA MiniKit kit or other kit, according to the kit instructions for extraction.
In a specific embodiment, the conditions of the multiplex fluorescent PCR amplification reaction are:
-pre-denaturation temperature: 94 ℃, pre-denaturation time: 5 minutes;
-45 cycles of:
Figure BDA0002487936640000072
and (3) denaturation temperature: 94 ℃, denaturation time: 30 seconds;
Figure BDA0002487936640000073
annealing temperature: 50 ℃, annealing time: 30 seconds;
Figure BDA0002487936640000074
extension temperature: 72 ℃, extension time: 1 minute and 30 seconds.
In yet another embodiment, the multiplex fluorescence PCR reaction system comprises 10 × ExTaq PCR buffer solution and 2.5mmol/L Mg2+0.25mmol/L dNTP, 1.5U Ex Taq polymerase, 0.05 mu mol/L LTR upstream primer and 0.05 mu mol/L LTR downstream primer; 0.05 u mol/L GAG upstream primer, 0.05 u mol/L GAG downstream primer, 0.05 u mol/L POL upstream primer, 0.05 u mol/L POL downstream primer, and 5.0L DNA template.
As described above, the multiplex fluorescent PCR amplification reaction may be performed using fluorescent probes. Therefore, in one embodiment of the method of the present invention, in a reaction system of 25 μ L of the multiplex fluorescence PCR, the following fluorescent probes are further included:
LTR fluorescent probe: 5'-FAM-CAAGCTAGTACCAGTT-ECLIPSE-3' (SEQ ID NO: 3);
GAG fluorescent probe: 5'-HEX-GAGTGCATCCAGTGCAT-ECLIPSE-3' (SEQ ID NO: 6);
POL fluorescent probe: 5'-CY5-ATTACAGGGACAGCAGAA-ECLIPSE-3' (SEQ ID NO: 9).
In addition, when ACTB is used as an internal reference, the method of the present invention may further comprise, in a reaction system of 25. mu.L of the multiplex fluorescence PCR:
ACTB upstream primer: 5'-ATTTGGACCTGCGAGCG-3' (SEQ ID NO: 10);
ACTB downstream primer: 5'-AGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 11); and
ACTB fluorescent probe: 5'-TEXASRED-CTGACCTGAAGGCTCTGC-ECLIPSE-3' (SEQ ID NO: 12).
Although the present inventors have described the reaction system of the present invention, it is understood that other conditions and steps of the multiplex fluorescent PCR amplification reaction can be modified or substituted according to actual needs by those skilled in the art, and such modifications and substitutions are within the scope of the present invention.
In a fourth aspect of the present invention, there is also provided a use of the primer pair of the first aspect of the present invention in the preparation of a detection agent for detecting an aids virus nucleic acid.
Optionally, in a fourth aspect of the invention, the primer pair is used in combination with the following fluorescent probes:
LTR fluorescent probe: 5'-FAM-CAAGCTAGTACCAGTT-ECLIPSE-3' (SEQ ID NO: 3);
GAG fluorescent probe: 5'-HEX-GAGTGCATCCAGTGCAT-ECLIPSE-3' (SEQ ID NO: 6);
POL fluorescent probe: 5'-CY5-ATTACAGGGACAGCAGAA-ECLIPSE-3' (SEQ ID NO: 9).
Further optionally, in a fourth aspect of the invention, the primer pair is also used in combination with:
ACTB upstream primer: 5'-ATTTGGACCTGCGAGCG-3' (SEQ ID NO: 10);
ACTB downstream primer: 5'-AGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 11); and
ACTB fluorescent probe: 5'-TEXASRED-CTGACCTGAAGGCTCTGC-ECLIPSE-3' (SEQ ID NO: 12).
The primer pair, the kit and the method of the invention realize simple, convenient and rapid detection of HIV virus nucleic acid, improve the sensitivity and specificity of detection, can directly detect the HIV virus nucleic acid from a whole blood sample or a dried blood spot sample, and the time from nucleic acid extraction to detection completion is only 3 hours, so the method is suitable for rapid diagnosis of HIV infection, in particular for early diagnosis of infection of exposed infants born by HIV positive puerperae, differential diagnosis of HIV antibody uncertain persons and detection of the HIV antibody in an antibody window period.
Examples
Hereinafter, the present invention will be described in more detail with reference to exemplary embodiments. However, the exemplary embodiments disclosed herein are for illustrative purposes only and should not be taken as limiting the scope of the invention.
Primers and probes: downloading gene sequences of HIV reference strains from American GenBank, performing homology comparison by using biological software, and designing specific primers and probes aiming at gene conserved regions, wherein the sequences are as follows:
LTR upstream primer: 5'-AAGGCTACTTCCCTGATT-3' (SEQ ID NO: 1);
LTR downstream primer: 5'-TTGGCTTCTTCTAACTTCTC-3' (SEQ ID NO: 2);
LTR fluorescent probe: 5'-FAM-CAAGCTAGTACCAGTT-ECLIPSE-3' (SEQ ID NO: 3);
GAG upstream primer: 5'-CAGAATGGGATAGAGTGC-3' (SEQ ID NO: 4);
GAG downstream primer: 5'-TACTGGGATAGGTGGATT-3' (SEQ ID NO: 5);
GAG fluorescent probe: 5'-HEX-GAGTGCATCCAGTGCAT-ECLIPSE-3' (SEQ ID NO: 6);
POL upstream primer: 5'-TAAGACAGCAGTACAAATGGCAG-3' (SEQ ID NO: 7);
POL downstream primer: 5'-GCTGTCCCTGTAATAAACCCG-3' (SEQ ID NO: 8);
POL fluorescent probe: 5'-CY5-ATTACAGGGACAGCAGAA-ECLIPSE-3' (SEQ ID NO: 9);
wherein FAM, HEX and CY5 are fluorescence reporter groups, and ECLIPSE is a fluorescence quencher group.
In addition, in the present application, β -actin is used as an internal reference against which the sequences of the corresponding primers and probes are:
ACTB upstream primer: 5'-ATTTGGACCTGCGAGCG-3' (SEQ ID NO: 10);
ACTB downstream primer: 5'-AGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 11);
ACTB fluorescent probe: 5'-TEXASRED-CTGACCTGAAGGCTCTGC-ECLIPSE-3' (SEQ ID NO: 12);
wherein TEXASRED is a fluorescence reporter group, and ECLIPSE is a fluorescence quencher group.
All the primers and probes were synthesized by Beijing Nonsui genome research center, Inc.
Optimizing a multiple fluorescence PCR reaction system and conditions: a Takara Ex Taq kit purchased from Dalibao bioengineering Co., Ltd was used to prepare a reaction system according to the instructions. For fluorescenceThe PCR reaction system mainly comprises MgCl2The concentration, the concentration of each primer and probe, the amount of Taq enzyme used, and the like are optimized, and the annealing temperature, time, cycle number, and the like are mainly optimized for the reaction program.
As a result, the following multiplex fluorescent PCR amplification reaction system and conditions were found to be suitable:
in a 25 mu L multiple fluorescence PCR amplification reaction system, the final concentration of each component is 10 × Ex Taq PCR buffer solution and 2.5mmol/L Mg2+0.25mmol/L dNTP, 1.5U Ex Taq polymerase, 0.05. mu. mol/L LTR upstream primer, 0.05. mu. mol/L LTR downstream primer, 0.05. mu. mol/L GAG upstream primer, 0.05. mu. mol/L GAG downstream primer, 0.05. mu. mol/LPOL upstream primer, 0.05. mu. mol/L POL downstream primer, 0.025. mu. mol/L ACTB upstream primer, 0.025. mu. mol/L ACTB downstream primer, 0.025. mu. mol/L LTR fluorescent probe, 0.25. mu. mol/L GAG fluorescent probe, 0.25. mu. mol/L POL fluorescent probe, 2.5. mu.L ACTB fluorescent probe, 5U Ex Taq, 5. mu.L DNA template;
the conditions of the multiplex fluorescence PCR amplification reaction are as follows:
-pre-denaturation temperature: 94 ℃, pre-denaturation time: 5 minutes;
-45 cycles of:
Figure BDA0002487936640000111
and (3) denaturation temperature: 94 ℃, denaturation time: 30 seconds;
Figure BDA0002487936640000112
annealing temperature: 50 ℃, annealing time: 30 seconds;
Figure BDA0002487936640000113
extension temperature: 72 ℃, extension time: 1 minute and 30 seconds.
And (5) judging a result: in the invention, for a certain gene in LTR, GAG and POL, the Ct value of the fluorescence PCR detection is less than 35, and the amplification curve is S-shaped and is positive, namely, the corresponding gene exists, and if no amplification curve or no Ct value is generated, the fluorescence PCR detection is judged to be negative, namely, the corresponding gene does not exist; as for the HIV-1 virus as a whole, if two or three of LTR, GAG and POL are positive, the existence of the HIV is judged, if none of the three is amplified, the existence of the HIV is judged, and if one is amplified positively, the existence of the HIV is judged to be uncertain.
Sensitivity test, using 8E5 cell diluted by multiple times as detection template, calculating and accurately quantifying its copy number, adding detection template 1 × 10 with corresponding copy number into the above multiple fluorescence PCR reaction system and conditions respectively6cps (copy number), 1 × 105cps、1×104cps、1×103cps, 100cps, 50cps, 10cps, 5cps and 0cps (nc) to examine the respective sensitivities of the LTR, GAG and POL sequences in the detection system under the aforementioned reaction conditions. All sensitivity experiments were set up with duplicate tubes to investigate the reproducibility of the detection system.
The results of the sensitivity test are shown in FIGS. 1 to 3, in which FIGS. 1 to 3 show the sensitivity results for the LTR, GAG and POL genes, respectively. As can be seen from the three figures, S-shaped amplification curves were obtained for all three genes with the addition of at least 10cps template. It can be seen that the lower limit of detection of the method of the present invention is as low as 10 cps.
And (3) detecting a clinical sample: the whole blood sample of HIV-infected person and the dried blood spot sample of the infant born by HIV-infected parturient are obtained from Yunnan, Xinjiang, Guangdong, Guangxi and Sichuan women's health care institute.
1. For adult whole blood samples: adult whole blood samples of 46 known healthy persons and 69 known HIV-1-infected persons (including 26 HIV-1CRF01_ AE recombinant strains, 13 HIV-1CRF07_ BC recombinant strains, 8 HIV-1CRF08_ BC recombinant strains, 14 HIV-1 subtype B strains, and 8 HIV-1 subtype C strains) were taken and used
Figure BDA0002487936640000121
After extracting DNA nucleic acid with the DNA Mini Kit (QIAGEN, Germany), the detection was performed by using the multiplex fluorescent PCR amplification reaction system, conditions and method.
The results showed that the blood samples of 46 known healthy persons were negative, and the blood samples of 69 known HIV-1 infected persons were positive. FIG. 4 shows typical results of detecting HIV-positive infected persons by the multiplex fluorescent PCR reaction system of the present invention, and FIG. 5 shows typical results of detecting HIV-negative infected persons by the multiplex fluorescent PCR reaction system of the present invention. As can be seen from FIG. 4, beta-action is efficiently amplified and detected, indicating that the detection system of the present invention is useful; and LTR, GAG and POL also all present S type amplification curve. In FIG. 5, however, only beta-action can be seen to be efficiently amplified and detected, but no S-type amplification curve appears in LTR, GAG and POL. The above results show that the method of the present invention can accurately distinguish healthy people from HIV-1 infected people without the occurrence of false positive and false negative phenomena. Furthermore, although the 69 HIV-1 infected individuals tested involved 5 subtypes of HIV-1 virus, they were all tested positive by the system of the present invention, confirming the ability of the method of the present invention to detect different types of HIV-1 strains.
Sample of dried blood spots from HIV-infected parturient born babies: tracking and follow-up 448 infants born by HIV-1 infected parturient women, collecting infant blood at 42 days old and 3 months old respectively to prepare dried blood spots, and using
Figure BDA0002487936640000131
The DNA Mini Kit (QIAGEN, Germany) extracts DNA nucleic acid, and then detects the DNA nucleic acid by adopting the multiplex fluorescence PCR amplification reaction system, the conditions and the method, and finally 432 effective results are obtained. Serum was isolated from infant venous blood at 18 months of age for antibody detection to verify the accuracy of its early virological diagnosis. Meanwhile, the inventor further adopts a commercial kit to carry out parallel tests to compare the effect of the invention relative to the prior art.
The results are shown in Table 1 below. It can be seen that, by using the multiplex fluorescence PCR amplification reaction system of the present invention, 26 positive infants were detected at 42 days of age, and the antibody was positively detected at 18 months of age; 403 negative infants were detected at 42 days of age and confirmed to be negative in the 18 months of age antibody test; there were 3 indeterminate infants at 42 days of age, which were negative by the detection kit of the present invention at 3 months of age for retest and confirmed to be negative in the antibody detection at 18 months of age. When the commercial kit is used, at 42 days of the infant, 1 case of the kit is judged to be false negative, and 5 cases of the kit are judged to be false positive.
Table 1: the PCR amplification system and the commercialized kit of the invention summarize the detection results of the dry blood spot samples of the infants born by the HIV-1 infected puerpera
Figure BDA0002487936640000132
Figure BDA0002487936640000141
From the above results, the reaction system of the present invention has extremely high sensitivity, and can perform effective and accurate detection even if the virus copy number in the sample is as low as 10 cps; the invention has no special choice for samples, and can detect whether the samples are adult whole blood samples or infant dry blood spot samples; in addition, the system can effectively and accurately detect HIV-1 of various subtypes, and is not only aimed at HIV-1 strains of a certain subtype.
In the present specification, whenever reference is made to "an exemplary embodiment", "a preferred embodiment", "one embodiment", or the like, it is intended that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. The appearances of such phrases in various places in the specification are not necessarily all referring to the same embodiment. Further, when a particular feature, structure, or characteristic is described in connection with any embodiment, it is submitted that it is within the purview of one skilled in the art to effect such feature, structure, or characteristic in other ones of all the embodiments described.
The embodiments of the present invention have been described above in detail. However, aspects of the present invention are not limited to the above-described embodiments. Various modifications and substitutions may be made to the above-described embodiments without departing from the scope of the invention.
Sequence listing
<120> reagent and method for qualitative detection of HIV
<160>12
<170>SIPOSequenceListing 1.0
<210>1
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>1
aaggctactt ccctgatt 18
<210>2
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>2
ttggcttctt ctaacttctc 20
<210>3
<211>16
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
caagctagta ccagtt 16
<210>4
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
cagaatggga tagagtgc 18
<210>5
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
tactgggata ggtggatt 18
<210>6
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
gagtgcatcc agtgcat 17
<210>7
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
taagacagca gtacaaatgg cag 23
<210>8
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
gctgtccctg taataaaccc g 21
<210>9
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
attacaggga cagcagaa 18
<210>10
<211>17
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
atttggacct gcgagcg 17
<210>11
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
agcggctgtc tccacaagt 19
<210>12
<211>18
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
ctgacctgaa ggctctgc 18

Claims (10)

1. A set of primer pairs for detecting hiv by multiplex fluorescent PCR amplification reactions, wherein the primer pairs comprise:
LTR upstream primer: 5'-AAGGCTACTTCCCTGATT-3' (SEQ ID NO: 1);
LTR downstream primer: 5'-TTGGCTTCTTCTAACTTCTC-3' (SEQ ID NO: 2);
GAG upstream primer: 5'-CAGAATGGGATAGAGTGC-3' (SEQ ID NO: 4);
GAG downstream primer: 5'-TACTGGGATAGGTGGATT-3' (SEQ ID NO: 5);
POL upstream primer: 5'-TAAGACAGCAGTACAAATGGCAG-3' (SEQ ID NO: 7);
POL downstream primer: 5'-GCTGTCCCTGTAATAAACCCG-3' (SEQ ID NO: 8).
2. The primer pair of claim 1, wherein the primer pair further comprises a primer pair for β -Actin (ACTB):
ACTB upstream primer: 5'-ATTTGGACCTGCGAGCG-3' (SEQ ID NO: 10);
ACTB downstream primer: 5'-AGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 11).
3. A kit for detecting hiv by multiplex fluorescent PCR amplification reactions, wherein said kit comprises: the primer set according to claim 1 or 2.
4. The kit of claim 3, wherein the kit further comprises the following fluorescent probes:
LTR fluorescent probe: 5'-FAM-CAAGCTAGTACCAGTT-ECLIPSE-3' (SEQ ID NO: 3);
GAG fluorescent probe: 5'-HEX-GAGTGCATCCAGTGCAT-ECLIPSE-3' (SEQ ID NO: 6);
POL fluorescent probe: 5'-CY5-ATTACAGGGACAGCAGAA-ECLIPSE-3' (SEQ ID NO: 9); and
optional ACTB fluorescent probe: 5'-TEXASRED-CTGACCTGAAGGCTCTGC-ECLIPSE-3' (SEQ ID NO: 12).
5. A method for detecting hiv by a multiplex fluorescent PCR amplification reaction for non-diagnostic purposes, comprising the steps of:
(1) extracting DNA of a sample to be detected;
(2) performing a multiplex fluorescence PCR amplification reaction using the primer set according to claim 1 or the kit according to claim 3, using a sample DNA to be tested as a template;
(3) judging the result of the multiplex fluorescence PCR amplification reaction:
if the Ct values of two or three of the three genes to be amplified are less than 35 and the amplification curve is S-shaped, then the existence of the AIDS virus is judged,
if none of the three amplified genes presents an amplification curve or a Ct value, determining that the HIV does not exist,
and if the Ct value of only one of the three amplified genes is less than 35 and the amplification curve is in an S type, determining that whether the AIDS virus exists or not.
6. The method of claim 5, wherein the conditions of the multiplex fluorescence PCR amplification reaction are:
-pre-denaturation temperature: 94 ℃, pre-denaturation time: 5 minutes;
-45 cycles of:
Figure FDA0002487936630000021
and (3) denaturation temperature: 94 ℃, denaturation time: 30 seconds;
Figure FDA0002487936630000022
annealing temperature: 50 ℃, annealing time: 30 seconds;
Figure FDA0002487936630000023
extension temperature: 72 ℃, extension time: 1 minute and 30 seconds.
7. The method of claim 5 or 6, wherein the reaction system of 25 μ L of the multiplex fluorescence PCR comprises 10 × Ex Taq PCR buffer and 2.5mmol/L Mg2+0.25mmol/L dNTP, 1.5U Ex Taq polymerase, 0.05 mu mol/L LTR upstream primer and 0.05 mu mol/L LTR downstream primer; 0.05. mu. mol/L GAG upstream primer, 0.05. mu. mol/LGAG downstream primer, 0.05. mu. mol/L POL upstream primer, 0.05. mu. mol/L POL downstream primer, and 5.0. mu.L DNA template.
8. The method of claim 7, wherein the reaction system of the multiplex fluorescence PCR comprises 25 μ L of the following fluorescent probes:
LTR fluorescent probe: 5'-FAM-CAAGCTAGTACCAGTT-ECLIPSE-3' (SEQ ID NO: 3);
GAG fluorescent probe: 5'-HEX-GAGTGCATCCAGTGCAT-ECLIPSE-3' (SEQ ID NO: 6);
POL fluorescent probe: 5'-CY5-ATTACAGGGACAGCAGAA-ECLIPSE-3' (SEQ ID NO: 9).
9. The method of claim 8, wherein in a reaction system of 25 μ L of the multiplex fluorescence PCR, further comprising:
ACTB upstream primer: 5'-ATTTGGACCTGCGAGCG-3' (SEQ ID NO: 10);
ACTB downstream primer: 5'-AGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 11); and
ACTB fluorescent probe:
5'-TEXASRED-CTGACCTGAAGGCTCTGC-ECLIPSE-3'(SEQ ID NO:12)。
10. use of the primer pair of claim 1 or 2 for the preparation of a detection agent for detecting an aids virus nucleic acid; optionally, the primer pair is used in combination with the following fluorescent probes:
LTR fluorescent probe: 5'-FAM-CAAGCTAGTACCAGTT-ECLIPSE-3' (SEQ ID NO: 3);
GAG fluorescent probe: 5'-HEX-GAGTGCATCCAGTGCAT-ECLIPSE-3' (SEQ ID NO: 6);
POL fluorescent probe: 5'-CY5-ATTACAGGGACAGCAGAA-ECLIPSE-3' (SEQ ID NO: 9);
further optionally, the primer pair is also used in combination with:
ACTB upstream primer: 5'-ATTTGGACCTGCGAGCG-3' (SEQ ID NO: 10);
ACTB downstream primer: 5'-AGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 11); and
ACTB fluorescent probe:
5'-TEXASRED-CTGACCTGAAGGCTCTGC-ECLIPSE-3'(SEQ ID NO:12)。
CN202010396986.4A 2020-05-12 2020-05-12 Reagent and method for qualitative detection of HIV Active CN111534640B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010396986.4A CN111534640B (en) 2020-05-12 2020-05-12 Reagent and method for qualitative detection of HIV

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010396986.4A CN111534640B (en) 2020-05-12 2020-05-12 Reagent and method for qualitative detection of HIV

Publications (2)

Publication Number Publication Date
CN111534640A true CN111534640A (en) 2020-08-14
CN111534640B CN111534640B (en) 2022-08-09

Family

ID=71973685

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010396986.4A Active CN111534640B (en) 2020-05-12 2020-05-12 Reagent and method for qualitative detection of HIV

Country Status (1)

Country Link
CN (1) CN111534640B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064923A (en) * 2022-01-27 2023-05-05 圣湘生物科技股份有限公司 Compositions, kits, methods and uses for detecting HIV-1

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106119413A (en) * 2016-07-01 2016-11-16 浙江省疾病预防控制中心 A kind of HIV (human immunodeficiency virus) multiple fluorescence PCR detection reagent box 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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106119413A (en) * 2016-07-01 2016-11-16 浙江省疾病预防控制中心 A kind of HIV (human immunodeficiency virus) multiple fluorescence PCR detection reagent box 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

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
FRANÇOIS ROUET,等: "In-house HIV-1 RNA real-time RT-PCR assays: Principle, available tests and usefulness in developing countries", 《EXPERT REV.MOL.DIAGN.》 *
MAURO S MALNATI，等: "A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load", 《NATURE PROTOCOLS》 *
吴守丽等: "多引物嵌套式聚合酶链反应在艾滋病病毒感染诊断中的应用", 《中国艾滋病性病》 *
魏民等: "人类免疫缺陷病毒1型感染基因诊断方法的建立", 《中华检验医学杂志》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116064923A (en) * 2022-01-27 2023-05-05 圣湘生物科技股份有限公司 Compositions, kits, methods and uses for detecting HIV-1

Also Published As

Publication number Publication date
CN111534640B (en) 2022-08-09

Similar Documents

Publication Publication Date Title
CN111235316B (en) Primer probe for identifying novel coronavirus and application of primer probe in triple fluorescence RPA
JP3689333B2 (en) Nucleic acid sequences that can be used as primers and probes to amplify and detect all subtypes of HIV-1
CN103074446B (en) Human immunodeficiency virus (HIV) nucleic acid detection kit
Nielsen et al. Diagnosis of HIV infection in children
CN109576397B (en) Human immunodeficiency virus type 1 nucleic acid quantitative detection kit
CN106119413A (en) A kind of HIV (human immunodeficiency virus) multiple fluorescence PCR detection reagent box and detection method
WO2014139330A1 (en) Rapid genotyping analysis and kits thereof
CN111286557A (en) Reagent combination, kit and application for detecting blood transfusion sexually transmitted pathogen
Mehta et al. Low-cost HIV-1 diagnosis and quantification in dried blood spots by real time PCR
Plantier et al. Plasma RNA quantification and HIV-1 divergent strains
Nakamichi et al. Evaluation of a quantitative real-time PCR assay for the detection of JC polyomavirus DNA in cerebrospinal fluid without nucleic acid extraction
WO2021254109A1 (en) Specific primer pair for hiv-1 viral load real-time fluorescence quantitative pcr test, and test kit
CN111534640B (en) Reagent and method for qualitative detection of HIV
Alvarez et al. Suppl 1: Improving Clinical Laboratory Efficiency: Introduction of Systems for the Diagnosis and Monitoring of HIV Infection
WO2020125295A1 (en) Primers, probes, kit and detection method for detecting human immunodeficiency virus nucleic acids
CN108998570B (en) HIV-1 total DNA quantitative detection primer pair, probe and detection kit capable of covering multiple subtypes
CN111500768B (en) Primer probe for identifying novel coronavirus and application of primer probe in dual-digital PCR
Khadir et al. Clinical evaluation of Amplicor HIV-1 test for detection of human immunodeficiency virus type 1 proviral DNA in peripheral blood mononuclear cells
Oladokun et al. Case Report: False-negative HIV-1 polymerase chain reaction in a 15-month-old boy with HIV-1 subtype C infection
JP2023517053A (en) CDI-enhanced COVID-19 testing
Jackson et al. Detection of human immunodeficiency virus type 1 (HIV-1) DNA and RNA sequences in HIV-1 antibody-positive blood donors in Uganda by the Roche AMPLICOR assay
US11459623B2 (en) In vitro method for detecting and quantifying HIV-2 DNA
CN107385116B (en) Method for detecting type 1 human immunodeficiency virus and special reagent set thereof
JP3351773B2 (en) HIV-1 subtype determination method
JP7182146B2 (en) Method and kit for measuring human T-cell leukemia virus type 1 (HTLV-1) proviral DNA

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant