CN113234853B

CN113234853B - Specific primer and kit for simultaneously detecting HIV-1, HBV, HCV and CMV

Info

Publication number: CN113234853B
Application number: CN202110401174.9A
Authority: CN
Inventors: 于斌; 姜淼
Original assignee: Beijing Liangxin Biotechnology Development Co ltd
Current assignee: Beijing Liangxin Biotechnology Development Co ltd
Priority date: 2021-04-14
Filing date: 2021-04-14
Publication date: 2022-05-13
Anticipated expiration: 2041-04-14
Also published as: CN113234853A

Abstract

The invention relates to the field of biomolecule detection, in particular to a specific primer and a kit for simultaneously detecting HIV-1, HBV, HCV and CMV. The invention provides a rapid, simple and convenient viral load detection kit for detecting HIV-1 virus and other three common concurrent infection viruses at one time by using a fluorescent quantitative PCR technology, and the kit has the advantages of good specificity of detection results, high sensitivity and accurate viral load detection results.

Description

Specific primer and kit for simultaneously detecting HIV-1, HBV, HCV and CMV

Technical Field

The invention relates to the field of biomolecule detection, in particular to a specific primer and a kit for simultaneously detecting HIV-1, HBV, HCV and CMV.

Background

Human Immunodeficiency Virus type i (HIV) is the main causative agent of Acquired Immunodeficiency Syndrome (AIDS) in humans, and is classified as a retrovirus (Retroviridae), Lentivirus (Lentivirus)

Hepatitis B Virus (HBV) is a pathogen causing Hepatitis B (Hepatitis B for short) and belongs to the hepadnaviridae. Among hepatitis B virus carriers, 50-75% of people have chronic hepatitis B with active virus replication, and the incidence rate of the progression from chronic hepatitis B to cirrhosis in 5 years is estimated to be 2-20%; from compensatory cirrhosis to liver decompensation of 20% -23%; the liver cancer is 6 to 15 percent from compensated liver cirrhosis. Chronic hepatitis b is the first risk factor for progression to cirrhosis, liver failure, and hepatocellular carcinoma. HBV is very contagious and 0.00004mL of virus-containing blood is inoculated sufficiently to infect humans. Hepatitis B virus often generates mutation, which brings great trouble to the accurate diagnosis and treatment of hepatitis B, so that an accurate and rapid laboratory test method is urgently needed to detect hepatitis B virus.

After infection with Hepatitis C Virus (HCV), there is usually a long window of antibody positive conversion, on average 70 days, some patients can be prolonged to 6-9 months or more, about 1-3% of patients can be continuously negative to anti-HCV, the replication of genome appears very early, and viremia appears several days after infection.

Cytomegalovirus (CMV) is a herpesvirus DNA virus, also known as a cell inclusion virus, and has a very broad infection among people, with a rate of adult infection in china of over 95%. Cytomegalovirus infection in normal populations is often latent, and latent CMV can be activated when the body is immunocompromised or inhibited.

In HIV-infected patients, HBV, HCV, CMV are frequently present in the peripheral blood of patients as co-infected viruses of HIV. Moreover, the existing clinical case tracking survey shows that HIV infected persons are more susceptible to concurrent virus infection compared with common persons because HBV and HCV are similar to the infection route of HIV in the infection route; in addition, the immune competence of HIV infected persons carrying concurrent infection viruses is more seriously reduced, and the HIV infected persons are more likely to progress to AIDS patients. Healthy people do not contain CMV, and patients often contain high-load CMV in the plasma of the patients in the later period of AIDS due to serious damage of the immune function of the patients with AIDS.

Disclosure of Invention

The purpose of the present application is to provide specific primers capable of simultaneously identifying HIV-1, HBV, HCV, CMV.

It is yet another object of the present application to provide a kit for multiplex fluorescent quantitative PCR viral load detection of HIV-1, HBV, HCV, CMV.

According to the application, specific primers capable of simultaneously identifying HIV-1, HBV, HCV and CMV are provided, wherein the HIV specific primers are as follows:

primer 1 HIVF: 5 '-AGTGGGGGGACAYCARGCAGC-3', and

primer 2 HIVR: 5 '-TACTAGTAGTTCCTGCTATRTCACTTCC-3';

the specific primers of HBV are:

primer 3 HBVF: 5'-GATGTGTCTGCGGCGTTTTA-3', and

primer 4 HBVR: 5'-GCAACATACCTTGATAGTCCAGAAGAA-3', respectively;

the HCV specific primers are:

primer 5 HCVF: 5'-AGCGTCTAGCCATGGCGTT-3', and

primer 6 HCVR: 5'-GCAAGCACCCTATCAGGCAGT-3', respectively;

the CMV-specific primers were:

primer 7 CMVF: 5'-TCAATCATGCGTTTGAAGAGGTA-3', and

primer 8 CMVR: 5'-ACCACCGCACTGAGGAATGTCAG-3' is added.

The multiple fluorescent quantitative PCR viral load detection kit for HIV-1, HBV, HCV and CMV comprises specific primers and probes for HIV-1, HBV, HCV and CMV, wherein,

specific primers and probes for HIV were:

primer 1 HIVF: 5 '-AGTGGGGGGACAYCARGCAGC-3' and

primer 2 HIVR: 5 '-TACTAGTAGTTCCTGCTATRTCACTTCC-3',

probe 1 HIVP: 5 '-FAM-AAGCTGCAGAATGGGATA-BHQ 1-3';

specific primers and probes for HBV were:

primer 3 HBVF: 5'-GATGTGTCTGCGGCGTTTTA-3' and

primer 4 HBVR: 5'-GCAACATACCTTGATAGTCCAGAAGAA-3' the flow of the air in the air conditioner,

probe 2 HBVP: 5 '-TET-CTTCCTCTTCATCCTGC-BHQ 1-3';

specific primers and probes for HCV were:

primer 5 HCVF: 5'-AGCGTCTAGCCATGGCGTT-3' and

primer 6 HCVR: 5'-GCAAGCACCCTATCAGGCAGT-3' the flow of the air in the air conditioner,

probe 3 HCVP: 5 '-JOE-TCCTTTCTTGGATAAACC-BHQ 1-3';

CMV specific primers and probes were:

primer 7 CMVF: 5'-TCAATCATGCGTTTGAAGAGGTA-3' and

primer 8 CMVR: 5'-ACCACCGCACTGAGGAATGTCAG-3'

Probe 4 CMVP: 5 '-NED-TCCACGTACTCGTAGGC-BHQ 1-3'.

The kit for detecting the multiple fluorescent quantitative PCR viral load of HIV-1, HBV, HCV and CMV comprises the following components: RT-PCR reaction buffer solution, primer probes, mixed enzyme, calibrator, positive reference substance and negative reference substance.

The embodiment of the application adopts at least one technical scheme which can achieve the following beneficial effects:

the invention provides a rapid, simple and convenient viral load detection kit for detecting HIV-1 virus and other three common concurrent infection viruses at one time by using a fluorescent quantitative PCR technology, and the kit has the advantages of good specificity of detection results, high sensitivity and accurate viral load detection results. The invention has another advantage that the patient can simultaneously detect 4 plasma viruses only by drawing blood once, thereby relieving the pain of the patient, avoiding the complex steps of multiple detections, shortening the detection time and saving the expenditure.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the application and together with the description serve to explain the application and not to limit the application. In the drawings:

FIG. 1 is an HIV positive sample amplification curve;

FIG. 2 is an HBV positive sample amplification curve;

FIG. 3 is an HCV positive sample amplification curve;

FIG. 4 is a CMV positive sample amplification curve;

FIG. 5 is a standard amplification curve, in which Δ Rn represents the fluorescence increment, i.e., the amplification product increment, and Ct value represents the cycle number;

FIG. 6 shows the preferred results of primer concentrations, where Δ Rn represents the fluorescence increment, i.e., the amplification product increment, and Ct values represent the cycle numbers;

FIG. 7 shows preferred results for probe concentration;

FIG. 8 is an amplification curve of an HIV positive sample detected in comparative example 1 using approximate primers of the present application;

FIG. 9 is an amplification curve of HBV positive samples detected using approximate primers of the present application in comparative example 1;

FIG. 10 is an amplification curve of HCV positive samples detected in comparative example 1 using approximate primers to the primers of the present application;

FIG. 11 is an amplification curve for detection of CMV positive samples using approximate primers of the present application in comparative example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail and completely with reference to the following specific embodiments of the present application and the accompanying drawings. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The technical solutions provided by the embodiments of the present application are described in detail below with reference to the accompanying drawings.

The invention designs 4 pairs of specific primers by inspecting genome sequences of Human Immunodeficiency Virus (HIV) I type, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) and Cytomegalovirus (CMV) and screening and optimizing. The optimal primers and probes are as follows:

primer 1 HIVF: 5 '-AGTGGGGGGACAYCARGCAGC-3'

Primer 2 HIVR: 5 '-TACTAGTAGTTCCTGCTATRTCACTTCC-3'

Probe 1 HIVP: 5 '-FAM-AAGCTGCAGAATGGGATA-BHQ 1-3'

Primer 3 HBVF: 5'-GATGTGTCTGCGGCGTTTTA-3'

Primer 4 HBVR: 5'-GCAACATACCTTGATAGTCCAGAAGAA-3'

Probe 2 HBVP: 5 '-TET-CTTCCTCTTCATCCTGC-BHQ 1-3'

Primer 5 HCVF: 5'-AGCGTCTAGCCATGGCGTT-3'

Primer 6 HCVR: 5'-GCAAGCACCCTATCAGGCAGT-3'

Probe 3 HCVP: 5 '-JOE-TCCTTTCTTGGATAAACC-BHQ 1-3'

Primer 7 CMVF: 5'-TCAATCATGCGTTTGAAGAGGTA-3'

Primer 8 CMVR: 5'-ACCACCGCACTGAGGAATGTCAG-3'

Probe 4 CMVP: 5 '-NED-TCCACGTACTCGTAGGC-BHQ 1-3'.

Wherein, the primer 1, the primer 2 and the probe 1 are used for detecting HIV-1, the primer 3, the primer 4 and the probe 2 are used for detecting HBV, the primer 5, the primer 6 and the probe 3 are used for detecting HCV, and the primer 7, the primer 8 and the probe 4 are used for detecting CMV.

The kit for detecting the multiple fluorescent quantitative PCR viral load of HIV-1, HBV, HCV and CMV comprises the specific primers and the fluorescent probes, and the kit is adopted to amplify target genes respectively by a real-time fluorescent quantitative RT-PCR method.

The nucleic acid of the sample to be detected can be extracted using a conventional phenol-chloroform extraction method or a commercially available nucleic acid extraction kit.

According to a specific embodiment of the invention, the kit comprises the following components:

the RT-PCR reaction solution was 30mM Tris-HCl (pH 8.3), 100mM KCl, 1.5mM dNTPs, and 4mM MgCl₂The mixed solution of (1); the enzyme mixture is a mixture of reverse transcriptase (3U/. mu.L) and TaqDNA polymerase (2U/. mu.L); the primers and probes provided were:

primers were 1.5. mu.M each;

The negative control substance is purified water; the positive control substance is artificially synthesized plasmid of HIV-1, HBV, HCV and CMV amplification target fragments with proper concentration; the 4 calibrators provided were dilutions of plasmids containing the four viral gene fragments, respectively, at known concentrations.

In the case where the other components in the reaction system were the same, the primer concentrations were serially diluted in multiples from 0.05. mu.M to 2.0. mu.M, respectively, and the results are shown in FIG. 6. By analytical comparison of the experimental results, the optimal primer concentration was determined to be 0.5. mu.M.

In the case where the other components in the reaction system were the same, the probe concentrations were each diluted in duplicate from 0.01. mu.M to 1.0. mu.M, and the results of the experiment are shown in FIG. 7. By analytical comparison of the experimental results, the optimum probe concentration was 0.05. mu.M.

The primers and the probes are used for establishing a reaction system, and finally, the optimal system of the fluorescent quantitative RT-PCR is determined to be 20 mu L.

The RT-PCR reaction conditions were optimized as follows: 30min at 48 ℃; 10min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 30s, 40 cycles. The fluorescence was collected at the "60 ℃ for 30 s" stage.

Example 1

1. Preparation of a sample template:

extracting virus nucleic acid from blood plasma by magnetic bead method, and storing the nucleic acid extract at-80 deg.C.

2. Preparing a reaction solution:

one step RT-PCR reaction buffer (final concentration) was prepared according to the following recipe:

30mM Tris-HCl(pH＝8.3)、100mM KCl、1.5mM dNTPs、4mM MgCl₂；

the mixed enzyme (final concentration) was prepared as follows:

reverse transcriptase (3U/. mu.L), TaqDNA polymerase (2U/. mu.L);

preparing a primer probe mixed solution (final concentration) according to the following formula:

primers (0.5. mu.M), probes (0.05. mu.M);

the calibrator was prepared as follows:

before use, the plasmids of the four viruses artificially synthesized were dissolved in ultrapure water and diluted to 10⁷copies/mL、10⁶copies/mL、10⁵copies/mL、10⁴copies/mL、10³copies/mL, in turn, as standards No. 1-5.

The kit set is as shown in table 1:

TABLE 1

Components (100 parts/box)	Volume of
		One step RT-PCR reaction buffer	1000μL(2×)
Primer 1+ primer 2+ Probe 1	250μL(4×)
		Primer 3+ primer 4+ Probe 2	250μL(4×)
Primer 5+ primer 6+ Probe 3	250μL(4×)
		Primer 7+ primer 8+ Probe 4	250μL(4×)
Mixed enzyme	250μL(4×)
		HIV-1 calibrator	Dry powder
HBV calibrator	Dry powder
		HCV calibrator	Dry powder
CMV calibrators	Dry powder

Preparing PCR reaction solution according to the formula of 10 mu L of One step RT-PCR reaction buffer solution, 2.5 mu L of mixed enzyme and 2.5 mu L of primer probe in each part, subpackaging the PCR reaction solution into 0.2mL of PCR tubes according to 15 mu L/tube, and then adding 5 mu L of nucleic acid extract to be detected as a template to ensure that the total reaction volume is 20 mu L.

PCR procedure:

the one-step RT-PCR assay was performed as follows: the reaction tube is firstly reacted at 48 ℃ for 30 minutes, then is insulated at 95 ℃ for 10 minutes, and is cycled for 40 times according to the temperature of 95 ℃ for 15 seconds → 60 ℃ for 30 seconds (the fluorescence of the corresponding probe labeling channel is collected under the annealing condition at 60 ℃).

4. The experimental results are as follows:

as shown in fig. 5: ct values of the standards 1-5<35 and linear correlation coefficient R²>0.99。

1) As shown in FIG. 1, the result of fluorescent quantitative PCR for detecting HIV shows that the Ct value of the sample is 32, and the negative control has no amplification.

2) As shown in FIG. 2, the result of fluorescence quantitative PCR for detecting HBV shows that the Ct value of the sample is 19, and the negative control has no amplification.

3) As shown in FIG. 3, the result of the fluorescent quantitative PCR for detecting HCV showed that the Ct value of the sample was 32 and that the negative control was not amplified.

4) As shown in FIG. 4, the result of the fluorescent quantitative PCR for CMV detection showed that the Ct value of the sample was 33 and that the negative control was not amplified.

The quantitative result of the corresponding virus analyzed by the instrument is directly read, the detection sensitivity of the primer and the probe provided by the invention is shown to reach 200copies/mL, and the detection rate is shown to be very good.

The primer and the probe provided by the invention have no signal when detecting a negative sample, and show good specificity.

Comparative example 1

For the nucleic acid extract obtained in example 1, other amounts were kept constant, and real-time fluorescent quantitative PCR amplification detection was performed using the following primers:

1.HIV-F AGTGGGGGGACAYCARGCAGC

2.HIV-R TACTAGTAGTTCCTGCTATRTCACTTCC

3.HBV-F TTCCGGAAACTACTGTTGTTAGAC

4.HBV-R ATTGAGATTCCCGAGATTGAGA

5.HCV-F CTGTCTTCACGCAGAAAGCG

6.HCV-R CACTCGCAAGCACCCTATCA

7.CMV-F GCGGTGGTTGCCCAACAGGA

8.CMV-R ACGACCCGTGGTCATCTTTA

the amplification results are shown in FIGS. 8-11, and the Ct values of the experimental groups are all greater than 35 or around 35. The amplification result of comparative example 1 shows that the amplification result of the application primer pair has no good sensitivity compared with the application primer pair, and the application primer pair is superior to the approximate primer pair.

Sequence listing

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Claims

1. A multiple fluorescence quantitative PCR viral load detection kit for HIV-1, HBV, HCV and CMV is characterized in that the kit comprises specific primers and probes for simultaneously detecting HIV-1, HBV, HCV and CMV, wherein,

specific primers and probes for detecting HIV were:

primer HIVF: 5 '-AGTGGGGGGACAYCARGCAGC-3' and

primer HIVR: 5 '-TACTAGTAGTTCCTGCTATRTCACTTCC-3',

and (3) probe HIVP: 5 '-FAM-AAGCTGCAGAATGGGATA-BHQ 1-3';

specific primers and probes for detecting HBV are as follows:

the primer HBVF: 5'-GATGTGTCTGCGGCGTTTTA-3' and

primer HBVR: 5'-GCAACATACCTTGATAGTCCAGAAGAA-3' the flow of the air in the air conditioner,

the probe HBVP: 5 '-TET-CTTCCTCTTCATCCTGC-BHQ 1-3';

specific primers and probes for detecting HCV were:

primer HCVF: 5'-AGCGTCTAGCCATGGCGTT-3' and

primer HCVR: 5'-GCAAGCACCCTATCAGGCAGT-3' the flow of the air in the air conditioner,

and (3) probe HCVP: 5 '-JOE-TCCTTTCTTGGATAAACC-BHQ 1-3';

the specific primers and probes for detecting CMV were:

primer CMVF: 5'-TCAATCATGCGTTTGAAGAGGTA-3' and

primer CMVR: 5'-ACCACCGCACTGAGGAATGTCAG-3'

And (3) probe CMVP: 5 '-NED-TCCACGTACTCGTAGGC-BHQ 1-3'.