Primer-probe sets, detection kits and detection methods for detecting HIV-1 and VB mutant strains

Abstract

The present invention discloses a primer-probe set, a detection kit and a detection method for detecting HIV-1 and VB mutant strains, belonging to the field of molecular biology detection. The primer-probe set includes primer-probe set A and primer-probe set B, wherein the primer-probe set A includes primers shown in SEQ ID NO: 1-2 and the probe gap-P shown in SEQ ID NO: 3; the primer-probe set B includes primers shown in SEQ ID NO: 4-5 and the probe gap-vb-P shown in SEQ ID NO: 6. By designing the primer-probe set, the present invention increases the detection targets, can effectively distinguish human immunodeficiency virus HIV-1 from VB mutant strains, and provides good technical support for the differential diagnosis of HIV-1 and VB mutant strains.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology detection, and particularly to a primer-probe set, a detection kit, and a detection method for detecting HIV-1 and VB mutant strains. Background Art

[0002] Human Immunodeficiency Virus (HIV) is the main pathogen that induces Acquired Immunodeficiency Syndrome (AIDS) in humans and belongs to the genus Lentivirus of the Retroviridae family in taxonomy. According to different genotypes, HIV can be divided into two types: HIV-1 and HIV-2. HIV-1 has four different groups: M, N, O, and P, each group caused by independent cross-species transmission. HIV-1 has a higher virulence, is more easily transmitted, and has also led to the vast majority of HIV infections globally.

[0003] The change in the viral load after HIV-1 infection is closely correlated with the pathogenesis of AIDS. The detection of the HIV-1 viral load can predict the occurrence, development, and prognosis of AIDS. Rapid and accurate detection is of great significance for disease monitoring, prevention of mother-to-child transmission, and observation of the efficacy of antiviral drugs. A paper in the journal Science in early 2022 pointed out that scientists at the Big Data Institute of the University of Oxford discovered a new, highly virulent strain of the HIV virus in the Netherlands. This brand-new strain belonging to the HIV-1 B' subtype was named the "VB mutant strain". Although the newly discovered AIDS virus variant is more virulent and more transmissible, after starting treatment, the immune system recovery and survival rate of VB variant patients are similar to those of other HIV variant patients. However, the researchers emphasized that since the VB mutant causes the immune system to decline more rapidly, it is crucial for individuals to be diagnosed as early as possible and start treatment as soon as possible. Therefore, there is an urgent need to develop a detection method for HIV-1 and VB mutant strains. Summary of the Invention

[0004] The object of the present invention is to provide a primer-probe set, a detection kit, and a detection method for detecting HIV-1 and VB mutant strains to solve the problems existing in the above-mentioned prior art. By designing the primer-probe set, the detection targets are increased, and it is possible to effectively distinguish between the human immunodeficiency virus HIV-1 and the VB mutant strain, providing good technical support for the differential diagnosis of HIV-1 and VB mutant strains.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a primer-probe set for detecting HIV-1 and VB mutant strains, including primer-probe set A and primer-probe set B. Among them, the primer-probe set A includes an upstream primer gap-F shown in SEQ ID NO: 1, a downstream primer gap-R shown in SEQ ID NO: 2, and a probe gap-P shown in SEQ ID NO: 3; the primer-probe set B includes an upstream primer gap-vb-F shown in SEQ ID NO: 4, a downstream primer gap-vb-R shown in SEQ ID NO: 5, and a probe gap-vb-P shown in SEQ ID NO: 6.

[0007] The present invention designs brand-new primer-probes that are completely different from the recommended standards through different sequence alignments. In actual sample tests, more positive samples are detected compared with the primer-probes in commercially available kits. The primer-probe set B of the gag gene, which is newly designed and optimized, is selected at the mutation site, which can avoid the formation of hairpin structures, primer internal dimers, primer-primer dimers, and mismatches as much as possible and can avoid non-specific binding to the HIV-1 wild type, other viruses, or human genes. The HIV-1 and its VB mutant strains can be distinguished using the same kit.

[0008] Preferably, in the above-mentioned primer-probe set for detecting HIV-1 and VB mutant strains, the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a fluorescent quenching group or an MGB quenching group. Probes for different target genes are labeled with different fluorescent groups respectively. This is convenient for detection using different fluorescence channels. More preferably, the fluorescent group can be selected from FAM, ROX, VIC, CY5, HEX; the quenching group is selected from BHQ1, BHQ2, BHQ3, MGB, and the excitation and emission bands of these groups are relatively separated, so they can be detected separately in one system. More preferably, in the above-mentioned primer-probe set for detecting HIV-1 and VB mutant strains, the probe gag-P is labeled with FAM, and the probe gag-vb-P is labeled with CY5.

[0009] The present invention also provides a kit for detecting HIV-1 and VB mutant strains, including the above-mentioned primer-probe set.

[0010] Preferably, the kit further includes: reaction premix reagent Premix Ex Taq, internal reference dye VIC Reference Dye II, positive control, and negative control. Among them, the positive control is standard plasmids pUC57-gag and pUC57-gag-vb; the negative control is double-distilled water.

[0011] Preferably, the gene copy number of the standard plasmid pUC57-gap is 2.9×10 9Copies / μL; the gene copy number of the standard plasmid pUC57-gag-vb is 2.5×10 9 Copies / μL.

[0012] The present invention also provides a method for detecting HIV-1 and VB mutant strains for non-disease diagnosis and treatment purposes, including performing fluorescence quantitative PCR detection on a test sample using the primer-probe set described above to generate an amplification curve, and analyzing the result of the amplification curve of the test sample. Adjust the Start value, End value, and Threshold value of Baseline according to the analyzed image (taking ABI 7500 as an example, the user can adjust according to the actual situation. The Start value can be between 3 and 15, and the End value can be set between 5 and 20 to ensure that the baseline part of all amplification curves is straight; the height of the threshold line for each fluorescence channel is set to △Rn = 6000), click Analyze for analysis, and then record the qualitative result under the Plate window. It also includes quality control: judging the experimental validity according to the Ct value corresponding to the detection result of each fluorescence channel and the morphology of the amplification curve.

[0013] Preferably, the reaction system for the fluorescence quantitative PCR detection includes: a mixture of 2 μL of the upstream primer gag-F, downstream primer gag-R, upstream primer gag-vb-F, and downstream primer gag-vb-R; a mixture of 1 μL of the probe gag-P and probe gag-vb-P; 12.5 μL of the reaction premix reagent PremixEx Taq; 0.2 μL of the internal reference dye VIC Reference Dye II; 2 μL of a mixture of the standard plasmids pUC57-gag and pUC57-gag-vb or 5 μL of the DNA template of the test sample; add double-distilled water to make up to 25 μL.

[0014] Preferably, the reaction program for the fluorescence quantitative PCR detection is: the reaction conditions of the reaction system are pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for 40 cycles.

[0015] The present invention also provides the application of the primer-probe set described above in the preparation of reagents or kits for detecting HIV-1 and VB mutant strains.

[0016] The present invention discloses the following technical effects:

[0017] The primer-probe set designed in the present invention is selected in the conserved region of the gene, and tries to avoid the formation of hairpin structures, internal primer dimers, inter-primer dimers and mismatches, and can avoid non-specific binding with other viruses or human genes. It can distinguish HIV-1 and its VB mutant strains with the same kit; the primer-probe set of the present invention can increase the detection targets, so as to effectively distinguish human immunodeficiency virus HIV-1 from VB mutant strains. It can be used for in vitro qualitative detection of HIV genes in samples such as blood, saliva, urine, etc. of suspected cases, suspected clustered cases, and other individuals who need to be diagnosed or differentially diagnosed for human immunodeficiency virus infection.

[0018] The detection method disclosed in the present invention has good specificity, sensitivity and repeatability, provides good technical support for the differential diagnosis of HIV-1 and VB mutant strains, and has good social application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 It is the amplification result diagram of the double fluorescence quantitative PCR of the HIV-1 wild type of the present invention;

[0021] Figure 2 It is the amplification result diagram of the double fluorescence quantitative PCR of the HIV-1 VB mutant strain of the present invention;

[0022] Figure 3 It is the standard curve of the double fluorescence quantitative PCR of the HIV-1 wild type and VB mutant strain of the present invention;

[0023] Figure 4 It is the detection result curve of the probe sensitivity of the double fluorescence quantitative PCR of the HIV-1 wild type of the present invention;

[0024] Figure 5 It is the amplification result diagram of the FAM probe sensitivity detection of the double fluorescence quantitative PCR of the HIV-1 VB mutant strain of the present invention;

[0025] Figure 6 It is the amplification result diagram of the CY5 probe sensitivity detection of the double fluorescence quantitative PCR of the HIV-1 VB mutant strain of the present invention;

[0026] Figure 7 It is the specific detection result of the double fluorescence quantitative PCR of the HIV-1 VB mutant strain of the present invention. Detailed implementation manners

[0027] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation on the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0028] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0030] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are only exemplary.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1 Primer design and condition optimization

[0033] According to the published HIV genome sequence in the NCBI GenBank database, the gag gene sequence of the classic strain K03455.1 of HXB2 in China was selected, and Primer3Plus (http: / / www.primer3plus.com) software was used for the design of primers and probes. The primers and probes were synthesized by Sangon Biotech (Shanghai) Co., Ltd., diluted to 10 μmol / L with sterile water, and stored at -20°C.

[0034] The primer-probe set A for the gag gene used to distinguish wild-type strains and mutant strains includes the primers gag-F and gag-R and the detection probe gag-P, and the sequences are as follows:

[0035] Forward primer gag-F (SEQ ID NO: 1): 5’-ATGTTAAAAGAGACCATCAATGAGG-3’;

[0036] Reverse primer gag-R (SEQ ID NO: 2): 5’-TCCTGCTATGTCACTTCCCCT-3’;

[0037] Probe gag-P (SEQ ID NO: 3): 5’-AGCTGCAGAATGGGA-3’.

[0038] The primer-probe set B includes the primers gag-vb-F and gag-vb-R and the detection probe gag-vb-P, and the sequences are as follows:

[0039] Forward primer gag-vb-F (SEQ ID NO: 4): 5’-GGGTGCGAGAGCGTCAGTAT-3’;

[0040] Reverse primer gag-vb-R (SEQ ID NO: 5): 5’-TGGCCGTAACCGAATTTTTT-3’;

[0041] Probe gag-vb-P (SEQ ID NO: 6): 5’-CGGCGGAAAATTAGACCA-3’.

[0042] Furthermore, the DNAstar Megalign software was used to evaluate the conservation of the sequences of the primers and probes in the prevalent strains. The primer and probe sequences designed in the present invention are highly conserved in the genotype 1 HIV strains prevalent in China and Eastern Europe.

[0043] Selection of the positive control in the kit in Example 2

[0044] The standard plasmids pUC57-gag and pUC57-gag-vb were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and stored in our laboratory. The concentration of the standard plasmids was measured using NanoDrop One (Thermo Fisher Scientific). The formula for calculating the gene copy number is as follows: Y (copies / μL) = [concentration of plasmid DNA (ng / μL) × 10-9 / (length of plasmid DNA bp × 660)] × 6.02 × 10 23 . After measurement, the gene copy numbers of the standard plasmids are 2.9 × 10 9copies / μL (pUC57 - gag) and 2.5×10 9 copies / μL (pUC57 - gag - vb).

[0045] Example 3 Establishment of Dual Fluorescent Quantitative PCR Detection Method

[0046] (1) Composition of the Dual Fluorescent Quantitative PCR Detection Kit

[0047] The dual fluorescent quantitative PCR detection kit includes a probe - based fluorescent quantitative detection reagent (Probe - based qPCR reaction Premix Ex Taq); internal reference dye VIC Reference Dye II; upstream primer gag - F, downstream primer gag - R, probe gag - P, upstream primer gag - vb - F, downstream primer gag - vb - R, probe gag - vb - P; positive control: standard plasmids pUC57 - gag and pUC57 - gag - vb; negative control: double - distilled water.

[0048] (2) Establishment of the Standard Curve

[0049] After preliminary screening, the reaction system of the dual fluorescent quantitative PCR was determined to be 25 μL, including 2 μL of the above - mentioned primer mixture, 1 μL of the probe mixture (the final concentration of each primer and probe was 0.2 μM, and the volume of each primer and probe was 0.5 μL), 12.5 μL of the probe - based qPCR reaction Premix Ex Taq, 0.2 μL of the internal reference dye VIC Reference Dye II, 2 μL of a 10 - fold serial dilution mixture of standard plasmids pUC57 - gag and pUC57 - gag - VB (the volume of each standard plasmid was 1 μL), and made up to 25 μL with double - distilled water.

[0050] The reaction conditions were pre - denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for 40 cycles. Fluorescent quantitative PCR detection was performed using the ABI7500Fast Real - Time PCR System, and a standard curve was generated. When the HIV - 1 wild - type plasmid pUC57 - gag was used as the template, an amplification curve appeared in a single detection channel ( Figure 1 ), when the HIV - 1 mutant plasmid pUC57 - gag - VB was used as the template, amplification curves appeared in both detection channels ( Figure 2 ).

[0051] As Figure 3 shown, when the template copy number was between 10 8 - 10 2When within the range, the established dual-fluorescent quantitative PCR detection method can obtain good amplification kinetic curves. The correlation coefficients of HIV-1 and HIV-VB are 0.999 and 0.998 respectively, and the efficiencies are 101.2 and 100.0 respectively, both within 90% - 110%, showing a good linear relationship; the regression equations of HIV-1 and HIV-VB are y = -3.293x + 42.809 and y = -3.321x + 41.490 respectively. Therefore, the clinical samples can be detected according to the Cq values of the detected clinical samples and with reference to the standard curve.

[0052] Example 5 Sensitivity Analysis Experiment

[0053] The pUC57-gag and pUC57-gag-VB recombinant plasmid standards were respectively diluted by 10-fold serial dilution. 2 μL was taken from each dilution group as the template for the dual-fluorescent quantitative PCR detection method, and amplified by the dual-fluorescent quantitative PCR established in the present invention.

[0054] As Figures 4 - 6 shown, when the concentrations of pUC57-gag and pUC57-gag-VB plasmids were 100 copies / μL, good specific amplification curves could still appear, indicating that the established method has high sensitivity. According to the results of 3 experimental repetitions, it was finally determined that when the Ct value ≤ 35, it could be judged as positive; when the Ct value ≥ 36, it was judged as negative; when 35 < Ct value < 36, it was judged as suspicious.

[0055] Example 6 Specificity Analysis Experiment

[0056] Using the cDNA or DNA of 8 kinds of infectious disease samples such as hepatitis A, hepatitis B, hepatitis C, syphilis, influenza, herpes, varicella, and rubella as templates, a positive control with pUC57-gag and pUC57-gag-VB standard plasmids as templates and a negative control with double-distilled water as the template were established simultaneously. Fluorescent quantitative PCR detection was carried out to evaluate the specificity of the established detection method.

[0057] As Figure 7 shown, specific amplification curves appeared in the positive control group with pUC57-gag and pUC57-gag-VB standard plasmids as templates, while no amplification curves appeared in the samples containing other pathogen cDNA or DNA, and the detections were all negative, indicating that the dual-fluorescent quantitative PCR detection method established in the present invention has good specificity.

[0058] Example 7 Repeatability Experiment

[0059] Seven consecutive dilutions of the standard plasmid were selected and detected in triplicate using the established duplex real-time PCR. Three replicates were set for each dilution within each batch, and the standard deviation and coefficient of variation within and between groups were calculated to evaluate the stability of the established detection method.

[0060] As shown in Table 1, the coefficients of variation within and between groups were both less than 5%. This indicates that the established real-time fluorescence detection method has good stability.

[0061] Table 1 Repeatability analysis of the duplex real-time PCR detection method

[0062]

[0063] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention. Sequence Listing <110> Zhengzhou University, Peking University <120> Primer-probe set, detection kit and detection method for detecting HIV-1 and VB mutant strains <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <400> 1 atgttaaaag agaccatcaa tgagg 25 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <400> 2 tcctgctatg tcacttcccc t 21 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <400> 3 agctgcagaa tggga 15 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <400> 4 gggtgcgaga gcgtcagtat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <400> 5 tggccgtaac cgaatttttt 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <400> 6 cggcggaaaa ttagacca 18

Claims

1. A primer-probe set for detecting HIV-1 and VB mutant strains, characterized in that, It includes primer-probe set A and primer-probe set B, wherein the primer-probe set A includes the upstream primer gap-F shown in SEQ ID NO: 1, the downstream primer gap-R shown in SEQ ID NO: 2, and the probe gap-P shown in SEQ ID NO: 3; the primer-probe set B includes the upstream primer gap-vb-F shown in SEQ ID NO: 4, the downstream primer gap-vb-R shown in SEQ ID NO: 5, and the probe gap-vb-P shown in SEQ ID NO: 6; The 5'-end of the probe gap-P and the probe gap-vb-P is labeled with a fluorescent group, and the 3'-end is labeled with a fluorescence quenching group; The fluorescent group includes FAM, ROX, VIC, CY5 or HEX; the fluorescence quenching group includes BHQ1, BHQ2 or BHQ3.

2. A kit for detecting HIV-1 and VB mutant strains, characterized in that, It includes the primer-probe set described in claim 1.

3. The kit according to claim 2, characterized in that, The kit further includes: reaction premix reagent PremixEx Taq, internal reference dye VIC Reference Dye II, positive control and negative control, wherein the positive control is the standard plasmids pUC57-gag and pUC57-gag-vb; the negative control is double-distilled water.

4. The kit according to claim 3, characterized in that, The gene copy number of the standard plasmid pUC57-gap is 2.9×10 9 copies / μL; the gene copy number of the standard plasmid pUC57-gag-vb is 2.5×10 9 copies / μL.

5. A method for detecting HIV-1 and VB mutant strains for non-disease diagnosis and treatment purposes, characterized in that, It includes using the primer-probe set described in claim 1 to perform fluorescence quantitative PCR detection on a test sample, generating an amplification curve, and analyzing the result of the amplification curve of the test sample.

6. The detection method according to claim 5, wherein, The reaction system of the fluorescence quantitative PCR detection includes: a mixture of the upstream primer gag-F, the downstream primer gag-R, the upstream primer gag-vb-F, and the downstream primer gag-vb-R in total of 2 μL; a mixture of the probe gag-P and the probe gag-vb-P in total of 1 μL; reaction premix reagent PremixEx Taq 12.5 μL; internal reference dye VIC Reference Dye II 0.2 μL; a mixture of the standard plasmids pUC57-gag and pUC57-gag-vb 2 μL or the DNA template of the test sample 5 μL; supplemented with double-distilled water to make up to 25 μL.

7. The detection method according to claim 5, characterized in that, The reaction program of the fluorescence quantitative PCR detection is: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 10 s, annealing at 60°C for 30 s, for 40 cycles.

8. Use of the primer-probe set described in claim 1 in the preparation of a reagent or kit for detecting HIV-1 and VB mutant strains.

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