CN107385116B

CN107385116B - Method for detecting type 1 human immunodeficiency virus and special reagent set thereof

Info

Publication number: CN107385116B
Application number: CN201710821700.0A
Authority: CN
Inventors: 乔阳; 余倩; 葛猛; 王宏伟
Original assignee: Beijing Genomeprecision Technology Co ltd
Current assignee: BEIJING GENOMEPRECISION TECHNOLOGY Co.,Ltd.
Priority date: 2017-09-13
Filing date: 2017-09-13
Publication date: 2021-02-19
Anticipated expiration: 2037-09-13
Also published as: CN107385116A

Abstract

The invention discloses a method for detecting type 1 human immunodeficiency virus and a special reagent set thereof. The kit comprises a primer pair HIV-1 and a probe HIV-1; the primer pair HIV-1 consists of a primer HIV-F and a primer HIV-R; the primer pair HIV-1 contains specific RNA segment in the target sequence of HIV-1 genome; the nucleotide sequence of the specific DNA fragment obtained by reverse transcription of the specific RNA fragment is shown as sequence 1 in the sequence table; the probe HIV-1 is a single-stranded DNA molecule consisting of 20-30 nucleotides, and is the same as a partial segment in a specific DNA fragment. Experiments prove that the kit is good in specificity and high in sensitivity (the sensitivity reaches 10 copies/reaction system) when used for detecting HIV-1. Meanwhile, the internal standard in the kit is used as a competitive internal standard of the target nucleic acid and can be used as a reference substance for preventing false negative results. The invention has important application value.

Description

Method for detecting type 1 human immunodeficiency virus and special reagent set thereof

Technical Field

The invention relates to the technical field of biology, in particular to a method for detecting human immunodeficiency virus type 1 and a special complete set of reagent thereof.

Background

Human Immunodeficiency Virus (HIV), an HIV Virus, is a lentivirus that infects cells of the Human immune system, a type of retrovirus. The virus can destroy or damage the function of the Immune system, cause progressive decline of the Immune system, and finally cannot play a role in resisting infection and diseases, so that various diseases and cancers can appear in a human body, namely Acquired Immune Deficiency Syndrome (AIDS), also called AIDS.

HIV can be phylogenetically divided into two types, human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2), and both can cause AIDS. HIV-1 is the first human retrovirus and is associated with a large percentage of HIV viral infections in the world, and its origin can be traced to simian immunodeficiency virus isolated from chimpanzee subspecies. HIV-2 is a second human retrovirus, but primarily restricted to Western Africa, originally derived from white-neck white-eyebrow monkeys. HIV-1 and HIV-2 are closely related and have 58%, 59% and 39% homology in the gag, pol and env genes, respectively. Typing of HIV is important for accurate diagnosis, detection and appropriate treatment.

The study showed that the HIV-1 source was classified into M, O, N, P subgroups. The subgroup M accounts for more than 90% of HIV infections worldwide. The M subgroup is further classified into A, B, C, D, F, G, H, J, K subtypes of 9 based on genetic differences, thus presenting a great challenge to the comprehensive diagnosis of HIV and the manufacture of effective vaccines. These subtypes have about 30% variation in the env gene and about 15% variation in the gag and pol genes. To date, over 50 subtypes of HIV have been discovered. Analysis of data for HIV-1 subtype showed that among all HIV-infected individuals worldwide, subtype B accounted for 56%, subtypes A, C, D, F, G accounted for 6.9%, 17.1%, 3.3%, 1.0%, and 1.0%, respectively, the three recombinant subtypes 01-AE, 02-AG, and 07-BC accounted for 6.0%, 2.6%, and 1.0%, respectively, while the other subtypes accounted for 5.5% in total. In HIV-infected individuals in China, the B subtype, C subtype and BC recombinant types account for 23.3%, 16.1% and 6.8%, respectively, the 01-AE, 07-BC and 08-BC account for 29.2%, 18.6% and 3.2%, respectively, and the other subtypes account for 2.8%. In 2015, 110 thousands of people die of AIDS-related diseases worldwide, and by the end of 2015, 3670 thousands of people are expected to infect AIDS viruses worldwide. Nowadays, AIDS is still a disease with extremely high harm to human beings, and is always highly valued by governments of various countries.

With the development of economic integration, the global population is in an unprecedented high-speed flow state, HIV subtypes and recombinant types are further increased and are irreversible, and the subtype distribution of HIV in various countries is rapidly changed along with the flow of HIV carriers. Although the research on HIV has been greatly advanced and the treatment level of AIDS is greatly improved, the current medical technology still has no thorough cure method for AIDS, so that the prevention and control of AIDS infection are currently important measures. AIDS, although highly contagious, has only three transmission routes, namely blood, maternal and sexual transmission. Although the AIDS is seriously damaged, the infection capacity of the AIDS is not particularly strong, and the harm of the AIDS to human can be greatly reduced as long as the infection is effectively prevented, so that the AIDS can be completely well prevented by blocking the transmission path. However, the precondition for preventing the disease in the transmission path is that the AIDS patient can be effectively diagnosed, and then the targeted preventive measures can be taken. Therefore, the AIDS virus infection can be confirmed and found early before symptoms appear, and the medicine can be taken early to control the harm of the AIDS virus to the immunity and improve the life quality and the life cycle of the AIDS virus infected person, which is of great importance.

Early detection of HIV was primarily carried out immunologically, by determining the presence or amount of relevant antibodies or antigens in a test sample. The immunological method for detecting HIV antibody is a basic detection method which is used in most countries, and the current status can not be replaced, and mainly comprises enzyme linked immunosorbent assay (ELISA), immunoblotting assay (WB) and the like. ELISA is the most main method for detecting HIV antibody, the detection reagent for primary screening has been developed to the fourth generation, and can simultaneously detect HIV antibody and P24 antigen, and a positive result can be detected about 2 weeks after infection. In addition, isolation and culture of viruses from infected individuals is also a reliable diagnostic method, but is often difficult or takes a long time to culture due to the low intracellular proliferation levels of the virus.

With the rapid development of related disciplines such as molecular biology, the nucleic acid detection technology is more and more emphasized, and becomes a common technology for detecting HIV in a laboratory. Nucleic acid is extracted from cells, tissues or body fluids of HIV-infected persons, i.e., RNA of HIV can be directly detected, and detection can be completely carried out before serological change. Currently, molecular biological detection methods commonly used for HIV mainly comprise RT-PCR detection, real-time fluorescent quantitative PCR (qRT-PCR) and the like. Because the detection technologies directly use the virus nucleic acid as a detection substrate, the detection sensitivity is greatly improved, the window period of HIV infection is obviously shortened, and the detection technology can monitor the detection process in real time and can carry out quantitative detection.

The TaqMan probe technique is a highly specific fluorescent quantitative PCR technique. The TaqMan probe is an oligonucleotide probe, a fluorescent group is connected to the 5 'end of the probe, and a quenching group is arranged at the 3' end of the probe. When the probe is intact or paired with the target sequence, the fluorescence emitted by the fluorophore is quenched due to proximity to the quencher at the 3' terminus, and no fluorescent signal is generated. During the PCR reaction extension process, the 5'→ 3' exonuclease activity of the Taq DNA polymerase degrades the probe, allowing the fluorophore to separate from the quencher, i.e., generating a fluorescent signal. After each PCR cycle, one molecule of amplification product is produced, accompanied by the generation of one molecule of fluorescent signal. As the number of amplification cycles increases, the released fluorophore signal accumulates, and the intensity of the fluorescent signal represents the copy number of the amplification product in an exponential synchronous growth process. The technology has the characteristics of strong specificity, high automation degree and the like, and effectively solves the problems of PCR pollution and the like. Some real-time fluorescence quantitative PCR based on TaqMan probe technology is used for HIV-1 detection kits on the market at present, but due to numerous HIV genotypes, the kits cannot cover all subtypes of HIV-1, and detection omission often exists.

Disclosure of Invention

The technical problem to be solved by the invention is how to detect HIV-1.

In order to solve the technical problems, the invention firstly provides a complete set of reagent for detecting HIV-1, which can comprise a primer pair HIV-1 and a probe HIV-1; the primer pair HIV-1 can be composed of a primer HIV-F and a primer HIV-R; the primer pair HIV-1 contains a specific RNA segment in a target sequence of the HIV-1 genome; the specific DNA fragment obtained by reverse transcription of the specific RNA fragment can be y1) or y2) as follows:

y1) single-stranded DNA molecule shown in sequence 1 in the sequence table;

y2) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1;

the probe HIV-1 can be a single-stranded DNA molecule consisting of 20-30 nucleotides, and is the same as a partial segment in the specific DNA segment.

The primer HIV-F can be a1) or a2) as follows:

a1) a single-stranded DNA molecule shown in a sequence 2 in a sequence table;

a2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 2 and having the same function as the sequence 2.

The primer HIV-R can be b1) or b2) as follows:

b1) a single-stranded DNA molecule shown in a sequence 3 in a sequence table;

b2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 3 and having the same function as the sequence 3.

The probe HIV-1 can be c1) or c2) as follows:

c1) a single-stranded DNA molecule shown in a sequence 4 in a sequence table;

c2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 4 and having the same function as the sequence 4.

The probe HIV-1 may have a fluorescent label at its end.

The two ends of the probe HIV-1 can be respectively marked with a fluorescence group and a quenching group.

The probe HIV-1 may have FAM fluorescent label at its 5 'end and TAMRA fluorescent label at its 3' end.

The kit specifically comprises the primer pair HIV-1 and the probe HIV-1.

In the kit, the molar ratio of the primer HIV-F, the primer HIV-R and the probe HIV-1 can be specifically 2.5: 2.5: 1.

in the above kit, the amounts of the primer HIV-F, the primer HIV-R and the probe HIV-1 may be as follows: 6.25. mu.M of the primer HIV-F, 6.25. mu.M of the primer HIV-R and 2.5. mu.M of the probe HIV-1.

Any of the kits described above may further comprise an internal standard and an internal standard detection probe; the internal standard is a single-stranded RNA molecule; the internal standard reverse transcription obtained DNA fragment can be z1) or z 2):

z1) single-stranded DNA molecule shown in sequence 5 of the sequence table;

z2) the DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 5 and has the same function as the sequence 5;

the internal standard detection probe can be a single-stranded DNA molecule consisting of 20-30 nucleotides, and is the same as a partial segment in a DNA fragment obtained by reverse transcription of the internal standard.

The internal standard detection probe can be d1) or d2) as follows:

d1) a single-stranded DNA molecule shown as a sequence 6 in a sequence table;

d2) and (b) a single-stranded DNA molecule obtained by substituting and/or deleting and/or adding one or more nucleotides to the sequence 6 and having the same function as the sequence 6.

The internal standard detection probe may have a fluorescent label at its end.

The two ends of the internal standard detection probe can respectively mark a fluorescence group and a quenching group.

The internal standard detection probe may have a JOE fluorescent label at the 5 'end and a TAMRA fluorescent label at the 3' end.

The kit specifically comprises the primer pair HIV-1, the probe HIV-1, the internal standard and the internal standard detection probe.

Any of the kits described above may further comprise a working standard; the working standard is a single-stranded RNA molecule; and the DNA fragment obtained by reverse transcription of the working standard is the specific DNA fragment.

Any of the kits described above may further comprise a negative control. The negative control may be normal human plasma without HIV-1 RNA.

Any of the kits described above may further comprise a reference. The reference substance can be 'human plasma containing HIV-1A subtype nucleic acid', 'human plasma containing HIV-1B subtype nucleic acid', 'human plasma containing HIV-1C subtype nucleic acid', 'human plasma containing HIV-1BC recombinant nucleic acid', 'human plasma containing HIV-1D subtype nucleic acid', 'human plasma containing HIV-1F1 subtype nucleic acid', 'human plasma containing HIV-1F2 subtype nucleic acid', "human plasma containing HIV-1G subtype nucleic acid", "human plasma containing HIV-1H subtype nucleic acid", "human plasma containing HIV-1J subtype nucleic acid", "human plasma containing HIV-1K subtype nucleic acid", "human plasma containing 01-AE recombinant nucleic acid", "human plasma containing 07-BC recombinant nucleic acid" or "human plasma containing 08-BC recombinant nucleic acid".

The kit specifically comprises the primer pair HIV-1, the probe HIV-1, the internal standard detection probe, the working standard, the negative control and the reference.

The method for preparing any one of the kit described above, comprising the step of separately packaging the primer HIV-F, the primer HIV-R and the probe HIV-1 in any one of the kit described above.

Kits containing any of the kits described above are also within the scope of the invention.

d1) Or d2) or d3) or d4) also belong to the scope of protection of the invention:

d1) use of a kit of any of the above in the manufacture of a kit for the detection of HIV-1;

d2) the use of any one of the above kits or the kit for detecting whether a sample to be tested contains or is suspected to contain HIV-1;

d3) use of a kit of any of the above in the manufacture of a kit for identifying HIV-1;

d4) use of any one of the above kits or the kit in the identification of whether a virus to be tested is or is preferably HIV-1.

The invention also provides a method for detecting whether a sample to be detected contains HIV-1, which can be A1) or A2):

A1) taking total RNA of a sample to be detected as a template, carrying out real-time quantitative PCR detection by using any one of the above-mentioned kit reagents, and then carrying out the following judgments: if the reagent set can realize real-time quantitative PCR of the total RNA, the sample to be detected contains or is suspected to contain HIV-1; if the reagent set can not realize real-time quantitative PCR of the total RNA, the sample to be detected does not contain or is suspected to contain HIV-1;

A2) detecting whether the DNA fragment obtained by the reverse transcription of the total RNA of the sample to be detected contains a specific DNA fragment, and then judging as follows: if the DNA fragment obtained by the reverse transcription of the total RNA of the sample to be detected contains the specific DNA fragment, the sample to be detected contains or is suspected to contain HIV-1; if the DNA fragment obtained by the reverse transcription of the total RNA of the sample to be detected does not contain the specific DNA fragment, the sample to be detected does not contain or is suspected to contain HIV-1;

the specific DNA fragment can be y1) or y2) as follows:

y1) single-stranded DNA molecule shown in sequence 1 in the sequence table;

y2) the DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in the sequence 1 and has the same function as the sequence 1.

The invention also provides a method for identifying whether the virus to be detected is HIV-1, which can be B1) or B2):

B1) taking RNA of a virus to be detected as a template, carrying out real-time quantitative PCR detection by using any one of the kit reagents, and then carrying out judgment as follows: if the kit can realize real-time quantitative PCR of the RNA, the virus to be detected is or is candidate to be HIV-1; if said kit does not enable real-time quantitative PCR of said RNA, then the virus to be tested is not or is not candidate for HIV-1;

B2) detecting whether the DNA fragment obtained by the reverse transcription of the RNA of the virus to be detected contains a specific DNA fragment, and then judging as follows: if the DNA fragment obtained by the reverse transcription of the RNA of the virus to be detected contains a specific DNA fragment, the virus to be detected is or is selected as HIV-1; if the DNA fragment obtained by the reverse transcription of the RNA of the virus to be detected does not contain the specific DNA fragment, the virus to be detected is not HIV-1 or the candidate is not HIV-1;

the specific DNA fragment can be y1) or y2) as follows:

y1) single-stranded DNA molecule shown in sequence 1 in the sequence table;

Above, "the kit may perform real-time quantitative PCR on the total RNA" or "the kit may perform real-time quantitative PCR on the RNA" may specifically be: the HIV-1 detection curve of the total RNA or the RNA is a positive amplification curve, the copy number of the HIV-1 is more than 10, and the internal standard detection curve is a positive amplification curve.

Above, "the kit is incapable of performing real-time quantitative PCR on the total RNA" or "the kit is incapable of performing real-time quantitative PCR on the RNA" may specifically be: the HIV-1 detection curve of the total RNA or the RNA is a negative amplification curve and the internal standard detection curve is a positive amplification curve.

In any of the above methods, the reaction system for performing real-time quantitative PCR detection may comprise: template, Tris-HCl buffer solution, KCl, dATP, dGTP, dCTP, dUTP, MgCl₂Reverse transcriptase, Taq DNA polymerase, UNG enzyme, primer pair HIV-1, probe HIV-1 and internal standard detection probe. The template can be total RNA of a sample to be detected or RNA of virus to be detected.

In any of the above methods, the reaction system for performing the real-time quantitative PCR detection may specifically comprise a template, Tris-HCl buffer, KCl, dATP, dGTP, dCTP, dUTP, and MgCl₂Reverse transcriptase, Taq DNA polymerase, UNG enzyme, primer pair HIV-1, probe HIV-1 and internal standard detection probe. The template can be total RNA of a sample to be detected or RNA of virus to be detected.

In any of the above methods, the reaction system for performing the "real-time quantitative PCR detection" may be specifically the system A. The 25. mu.L system A consisted of 10. mu.L of template and 15. mu.L of RT-PCR reaction. The template can be total RNA of a sample to be detected or RNA of virus to be detected.

The RT-PCR reaction solution may contain 100mM KCl, 0.4mM dATP, 0.4mM dGTP, 0.4mM dCTP, 0.4mM dUTP, 6mM MgCl₂3U/. mu.L reverse transcriptase, 2U/. mu.L Taq DNA polymerase, 1U/. mu.L UNG enzyme, 6.25. mu.M primer HIV-F, 6.25. mu.M primer HIV-R, 2.5. mu.M probe HIV-1 and 2.5. mu.M internal standard detection probe pH8.3, 20mM Tris-HCl buffer.

In any of the above methods, the reaction procedure of "performing real-time quantitative PCR detection" may specifically be: 10min at 37 ℃; 15min at 50 ℃; 2min at 95 ℃; 94 ℃ 10sec, 60 ℃ 45sec, 40 cycles.

As above, the sample to be tested may be plasma to be tested.

Experiments prove that the kit provided by the invention has good specificity and higher sensitivity (the sensitivity reaches 10 copies/reaction system) when used for detecting HIV-1. Meanwhile, in the kit provided by the invention, the internal standard is taken as a competitive internal standard of the target nucleic acid and can be taken as a reference object for preventing false negative results, and whether the whole real-time quantitative PCR detection system is inhibited or not can be known by detecting the sample added with the internal standard, so that false negative can be prompted conveniently. The invention has important application value.

Drawings

FIG. 1 shows the results of the sensitivity test.

FIG. 2 shows the experimental results of the detection of the reference substance.

FIG. 3 shows the results of the specificity experiment.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The QIAamp Viral RNA Mini Kit is a product of QIAGEN. The high-purity plasmid large-extraction kit is a product of Tiangen Biochemical technology (Beijing) Co., Ltd, and the catalog number of the product is DP 116. The vector pGEM-T is a product of Promega corporation, and the product number is A362A. Both T7RNA polymerase and DNase I are products of Takara, having product numbers 2540 and 2270, respectively.

EXAMPLE 1 preparation of kit for detecting HIV-1

First, preparation of RT-PCR reaction solution

Through a large number of experiments, the inventor screens a conserved sequence with the length of 209bp (the transcribed nucleotide sequence is shown as a sequence 1 in a sequence table) by comparing and analyzing the nucleotide sequences of various subtypes of HIV-1. According to the conserved sequence, a primer HIV-F, a primer HIV-R, a probe HIV-1 and an internal standard detection probe are designed and synthesized. In the nucleotide sequences of the primers and probes, H ═ a/T/C, R ═ a/G, K ═ T/G, Y ═ C/T, and M ═ a/C.

The RT-PCR reaction solution consists of Tris-HCl buffer solution, KCl, dATP, dGTP, dCTP, dUTP and MgCl₂Reverse transcriptase, Taq DNA polymerase, UNG enzyme, primer pair HIV-1 (consisting of primer HIV-F and primer HIV-R), probe HIV-1 and internal standard detection probe.

The RT-PCR reaction solution contained 100mM KCl, 0.4mM dATP, 0.4mM dGTP, 0.4mM dCTP, 0.4mM dUTP, and 6mM MgCl₂3U/. mu.L reverse transcriptase, 2U/. mu.L Taq DNA polymerase, 1U/. mu.L UNG enzyme, 6.25. mu.M primer HIV-F, 6.25. mu.M primer HIV-R, 2.5. mu.M probe HIV-1 and 2.5. mu.M internal standard detection probe pH8.3, 20mM Tris-HCl buffer.

The primer HIV-F: 5 '-CGAHATAAARGTAGTRCCAAGAAG-3' (SEQ ID NO: 2 in the sequence listing).

The primer HIV-R: 5 '-CCATGTKYTAATCCTCATCCT-3' (SEQ ID NO: 3 in the sequence listing).

The probe HIV-1: 5 '-FAM-ATGGCAGGTGMTGATTGTRTGGCA-TAMRA-3' (SEQ ID NO: 4 in the sequence listing).

Internal standard detection probe: 5 '-JOE-TCTCCAATACCTTATCCCTCGCCTCC-TAMRA-3' (SEQ ID NO: 6 in the sequence listing).

The 5 'end of the probe HIV-1 has FAM fluorescent label, and the 3' end has TAMRA fluorescent label.

The internal standard detection probe has a JOE fluorescent label at the 5 'end and a TAMRA fluorescent label at the 3' end.

Preparation of complete set of reagent for detecting HIV-1

The kit for detecting HIV-1 consists of RT-PCR reaction liquid, a working standard, a negative control, an internal standard and a reference substance.

1. Working standard

(1) Synthesizing the double-stranded DNA molecule shown in the sequence 1 in the sequence table by adopting a chemical synthesis method.

(2) And (2) connecting the double-stranded DNA molecule synthesized in the step (1) with a vector pGEM-T to obtain a recombinant plasmid.

(3) And (3) introducing the recombinant plasmid obtained in the step (2) into escherichia coli DH5 alpha to obtain recombinant escherichia coli.

(4) The recombinant Escherichia coli obtained in step (3) was inoculated into 5mL of LB medium containing 100. mu.g/mL ampicillin, followed by shaking culture at 37 ℃ and 190rpm for 12 hours to obtain a bacterial solution.

(5) And (5) taking the bacterial liquid obtained in the step (4), and extracting plasmids according to the operation steps of the high-purity plasmid large-scale extraction kit.

(6) And (5) taking the plasmid obtained in the step (5), firstly carrying out in-vitro transcription by adopting T7RNA polymerase, and then digesting by adopting DNase I to remove DNA so as to obtain an RNA solution.

Diluting the RNA solution obtained in the step (6) with water without ribonuclease to obtain the RNA copy number of 10⁴copies/. mu.L RNA solution 1, RNA copy number 10³copies/. mu.L RNA solution 2, RNA copy number 10²copies/. mu.L RNA solution 3, RNA copy number 10¹copies/. mu.L RNA solution 4 and RNA copy number 10⁰copies/. mu.L RNA solution 5.

The RNA solution 1, the RNA solution 2, the RNA solution 3, the RNA solution 4 and the RNA solution 5 are working standards.

2. Negative control

The negative control was normal human plasma without HIV-1 RNA.

3. Internal standard

(1) A chemical synthesis method is adopted to synthesize the double-stranded DNA molecule shown in the sequence 5 in the sequence table (the only difference between the sequence 5 and the sequence 1 is that the nucleotide sequences of the internal standard detection probe regions are different).

(6) And (3) taking the plasmid obtained in the step (5), firstly carrying out in-vitro transcription by adopting T7RNA polymerase, and then digesting by adopting DNase I to remove DNA, so as to obtain an RNA solution.

Diluting the RNA solution obtained in the step (6) with water without ribonuclease to obtain the RNA copy number of 10⁵Internal standards of copies/. mu.L.

It should be noted that the internal standard is used as a competitive internal standard of the target nucleic acid and can be used as a reference substance for preventing false negative results, and whether the whole real-time quantitative PCR detection system is inhibited or not can be known by detecting the sample added with the internal standard, so that false negative can be prompted conveniently.

4. Reference article

Human plasma containing HIV-1 subtype A nucleic acid, human plasma containing HIV-1 subtype B nucleic acid, human plasma containing HIV-1 subtype C nucleic acid, human plasma containing HIV-1BC recombinant nucleic acid, human plasma containing HIV-1 subtype D nucleic acid, human plasma containing HIV-1F1 subtype nucleic acid, human plasma containing HIV-1F2 subtype nucleic acid, human plasma containing HIV-1G subtype nucleic acid, human plasma containing HIV-1H subtype nucleic acid, human plasma containing HIV-1J subtype nucleic acid, human plasma containing HIV-1K subtype nucleic acid, human plasma containing 01-AE recombinant nucleic acid, human plasma containing 07-BC recombinant nucleic acid and human plasma containing 08-BC recombinant nucleic acid are all provided by the general military hospitals, and the providers agree.

Example 2 establishment of a method for detecting HIV-1

First, extraction of total RNA of blood plasma to be tested

(1) And adding 10 mu L of internal standard into the blood plasma to be detected, and uniformly mixing to obtain a mixed solution.

(2) Taking the mixed solution, extracting RNA according to the operation steps of a QIAamp Viral RNA Mini Kit, and obtaining the total RNA of the plasma to be detected with the concentration of 10 ng/mu L.

Secondly, preparing a reaction system

The reaction system A is 25 mu L and consists of 10 mu L of total RNA of plasma to be detected and 15 mu L of RT-PCR reaction liquid.

Reaction system B (negative control) was 25. mu.L, consisting of 10. mu.L of total RNA from normal plasma and 15. mu.L of RT-PCR reaction. The steps for preparing the total RNA of the normal plasma are as follows: taking normal human plasma without HIV-1RNA, adding 10 mu L of internal standard, and uniformly mixing to obtain a mixed solution; the mixture was subjected to RNA extraction according to the procedure of QIAamp Viral RNA Mini Kit to obtain total RNA of normal plasma at a concentration of 10 ng/. mu.L.

The reaction system C (reference) was 25. mu.L, and consisted of 10. mu.L of total RNA of positive plasma (total plasma RNA containing HIV-1 subtype A RNA, total plasma RNA containing HIV-1 subtype B RNA, total plasma RNA containing HIV-1 subtype C RNA, total plasma RNA containing HIV-1 subtype BC RNA, total plasma RNA containing HIV-1 subtype D RNA, total plasma RNA containing HIV-1F1 subtype RNA, total plasma RNA containing HIV-1F2 subtype RNA, total plasma RNA containing HIV-1G subtype RNA, total plasma RNA containing HIV-1H subtype RNA, total plasma RNA containing HIV-1J subtype RNA, total plasma RNA containing HIV-1K subtype RNA, total plasma RNA containing HIV-101-AE subtype RNA, total plasma RNA containing HIV-107-BC RNA or total plasma RNA containing HIV-108-BC RNA) and 15. mu.L of RT-PCR reaction solution.

The steps for preparing HIV-1 subtype A RNA are as follows: taking human plasma containing HIV-1A subtype nucleic acid, adding 10 mu L of internal standard, and uniformly mixing to obtain a mixed solution; taking the mixed solution, extracting RNA according to the operation steps of a QIAamp Viral RNA Mini Kit, and diluting to obtain the total plasma RNA with the concentration of 10 ng/mu L and containing HIV-1A subtype RNA.

According to the above steps, the "human plasma containing HIV-1 subtype A nucleic acid" is respectively replaced by "human plasma containing HIV-1 subtype B nucleic acid", "human plasma containing HIV-1 subtype C nucleic acid", "human plasma containing HIV-1BC recombinant nucleic acid", "human plasma containing HIV-1 subtype D nucleic acid", "human plasma containing HIV-1F1 subtype nucleic acid", "human plasma containing HIV-1F2 subtype nucleic acid", "human plasma containing HIV-1G subtype nucleic acid", "human plasma containing HIV-1H subtype nucleic acid", "human plasma containing HIV-1J subtype nucleic acid", "human plasma containing HIV-1K subtype nucleic acid", "human plasma containing 01-AE recombinant nucleic acid", "human plasma containing 07-BC recombinant nucleic acid" and "human plasma containing 08-BC recombinant nucleic acid", the other steps are not changed, and the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1B subtype RNA, the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1C subtype RNA, the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1BC subtype RNA, the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1D subtype RNA, the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1F1 subtype RNA, the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1F2 subtype RNA, the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1G subtype RNA, the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1H subtype RNA, the total plasma RNA with the concentration of 10 ng/mu L and the HIV-1J subtype RNA, the total plasma RNA and the RNA and, Total plasma RNA containing HIV-1K subtype RNA at a concentration of 10 ng/. mu.L, total plasma RNA containing HIV-101-AE subtype RNA at a concentration of 10 ng/. mu.L, total plasma RNA containing HIV-107-BC subtype RNA at a concentration of 10 ng/. mu.L, and total plasma RNA containing HIV-108-BC subtype RNA at a concentration of 10 ng/. mu.L.

The reaction system D (working standard) is 25 mul, and consists of 10 mul of working standard (RNA solution 1, RNA solution 2, RNA solution 3, RNA solution 4 or RNA solution 5) and 15 mul of RT-PCR reaction solution.

Third, real-time quantitative PCR detection

And (4) carrying out real-time quantitative PCR detection on each reaction system prepared in the step two on a real-time fluorescent PCR instrument. Reaction conditions are as follows: 10min at 37 ℃; 15min at 50 ℃; 2min at 95 ℃; 94 ℃ 10sec, 60 ℃ 45sec, 40 cycles. Fluorescence signals of FAM channel and JOE channel were collected at 60 ℃. Wherein, FAM channel detects HIV-1, JOE channel detects the interior label.

Fourthly, judging the result

And after the third step is finished, adjusting the threshold line to be above the background signal and the negative control amplification line, and automatically calculating the copy number of the HIV-1 in each sample by the system according to the standard curve prepared by the reaction system and the CT value. Then, the following judgment is made:

1. if the HIV-1 detection curve of the total RNA of the plasma to be detected is a negative amplification curve and the internal standard detection curve is a positive amplification curve, the plasma to be detected does not contain HIV-1 (namely true negative);

2. if the HIV-1 detection curve of the total RNA of the plasma to be detected is a positive amplification curve, the copy number of the HIV-1 is more than 10 and the internal standard detection curve is the positive amplification curve, the plasma to be detected contains or is suspected to contain the HIV-1;

3. if the HIV-1 detection curve of the total RNA of the plasma to be detected is a positive amplification curve, the copy number of the HIV-1 is less than 10, and the internal standard detection curve is the positive amplification curve, HIV-1 nucleic acid pollution exists in the total RNA of the plasma to be detected, and the re-detection is needed;

4. if the internal standard detection curve is a negative amplification curve, inhibition exists in total RNA of the plasma to be detected or the PCR amplification reaction is abnormal, and the re-detection is needed.

The positive amplification curve is the "S-type" amplification curve. Negative amplification curves are not "sigmoidal" amplification curves.

In the negative control, the HIV-1 detection curve is a negative amplification curve and the internal standard detection curve is a positive amplification curve.

In the reference, the HIV-1 detection curve is a positive amplification curve and the internal standard detection curve is a positive amplification curve.

Example 3 sensitivity test

Firstly, preparing a reaction system

The reaction system a was 25. mu.L, and consisted of 10. mu.L of RNA solution (RNA solution 1, RNA solution 2, RNA solution 3, RNA solution 4, or RNA solution 5) and 15. mu.L of RT-PCR reaction solution.

Reaction System B the same reaction System B as in step two of example 2.

Reaction system C the same as in step two of example 2.

The reaction system D was the same as that in the second step of example 2.

Second, real-time quantitative PCR detection

The same procedure as in step three of example 2.

Thirdly, judging the result

The same procedure as in step four of example 2.

The HIV-1 detection curve is shown in FIG. 1(1 is

RNA solution

1, 2 is

RNA solution

2, 3 is

RNA solution

3, 4 is

RNA solution

4, and 5 is RNA solution 5). The results show that the sensitivity of the kit for detecting HIV-1 provided in example 1 is 10 copies/reaction system.

Example 4 detection experiment of reference substance

Firstly, preparing a reaction system

Reaction system B was 25. mu.L, and consisted of 10. mu.L of total RNA of positive plasma (total plasma RNA containing HIV-1 subtype A RNA, total plasma RNA containing HIV-1 subtype B RNA, total plasma RNA containing HIV-1 subtype C RNA, total plasma RNA containing HIV-1 subtype BC RNA, total plasma RNA containing HIV-1 subtype D RNA, total plasma RNA containing HIV-1F1 subtype RNA, total plasma RNA containing HIV-1F2 subtype RNA, total plasma RNA containing HIV-1G subtype RNA, total plasma RNA containing HIV-1H subtype RNA, total plasma RNA containing HIV-1J subtype RNA, total plasma RNA containing HIV-1K subtype RNA, total plasma RNA containing HIV-101-AE subtype RNA, total plasma RNA containing HIV-107-BC subtype RNA, or total plasma RNA containing HIV-108-BC subtype RNA in example 2, step two) and 15. mu.L of RT-PCR reaction solution.

Reaction System B the same reaction System B as in step two of example 2.

The reaction system D was the same as that in the second step of example 2.

Second, real-time quantitative PCR detection

The same procedure as in step three of example 2.

Thirdly, judging the result

The same procedure as in step four of example 2.

A part of HIV-1 detection curve is shown in FIG. 2(A is total plasma RNA containing HIV-1 subtype A RNA, B is total plasma RNA containing HIV-1 subtype B RNA, C is total plasma RNA containing HIV-1 subtype C RNA, and BC is total plasma RNA containing HIV-1 subtype BC RNA). The results show that the set of reagents provided in example 1 can be used to detect common subtypes of HIV-1 (e.g., subtype A, subtype B, subtype C, subtype BC, subtype D, subtype F1, subtype F2, subtype G, subtype H, subtype J, subtype K, subtype 01-AE, subtype 07-BC, and subtype 08-BC).

Example 5 specificity test

Firstly, preparing a reaction system

The reaction system c is 25 μ L, and consists of 10 μ L of total RNA of a sample to be tested (total RNA of HIV-1, total RNA of HTLV, total RNA of HCV, total RNA of influenza virus, total RNA of measles virus, total RNA of HBV, total RNA of HPV or total RNA of herpes simplex virus) and 15 μ L of RT-PCR reaction solution. Strains of HIV-1, HTLV, HCV, influenza virus, measles virus, HBV, HPV and herpes simplex virus are all provided by the national people Release military 302 Hospital.

Reaction System B the same reaction System B as in step two of example 2.

Reaction system C the same as in step two of example 2.

The reaction system D was the same as that in the second step of example 2.

Second, real-time quantitative PCR detection

The same procedure as in step three of example 2.

Thirdly, judging the result

The same procedure as in step four of example 2.

A partial HIV-1 detection curve is shown in FIG. 3 (other viruses are HTLV, HCV, influenza virus, measles virus, HBV, HPV and herpes simplex virus). The results show that the kit provided in example 1 has high specificity for detecting HIV-1.

Example 6 application

First, obtaining total RNA of sample to be tested

The total amount of the samples to be tested is 30. The test samples 1 to 28 were 2mL of venous blood of a human identified as containing HIV-1 in plasma. The test sample 29 and the test sample 30 were 2mL of venous blood of a human identified as having no HIV-1 in plasma.

1. Respectively adding samples 1 to 30 to be detected into a sterile collecting pipe containing EDTA or sodium citrate anticoagulant, slightly inverting and uniformly mixing, and centrifuging at room temperature and 2000rpm for 5 min.

2. After completion of step 1, the upper plasma was collected and the pellet was transferred to another sterile centrifuge tube.

3. And (3) after the step 2 is finished, adding 10 mu L of internal standard into the sterile centrifuge tube, and uniformly mixing to obtain a mixed solution.

4. And (3) after the step 3 is finished, taking the mixed solution, and extracting RNA according to the operation steps of the QIAamp Viral RNA Mini Kit to obtain the total RNA of the sample to be detected.

Secondly, preparing a reaction system

The reaction system d is 25 mul, and consists of 10 mul total RNA of the sample to be detected and 15 mul RT-PCR reaction liquid.

Reaction System B the same reaction System B as in step two of example 2.

Reaction system C the same as in step two of example 2.

The reaction system D was the same as that in the second step of example 2.

Third, real-time quantitative PCR detection

The same procedure as in step three of example 2.

Fourthly, judging the result

The same procedure as in step four of example 2.

The result shows that the samples 1 to 28 to be detected are all identified as containing HIV-1 in the plasma, and the samples 29 to be detected and the samples 30 to be detected are all identified as containing no HIV-1 in the plasma, and the results are completely consistent with the actual effect.

The results show that the kit provided in example 1 is used for detecting the sample to be detected containing HIV-1, and the results are accurate and reliable.

<110> Beijing FuanHua Biotech Co., Ltd

<120> method for detecting type 1 human immunodeficiency virus and special reagent set thereof

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 209

<212> DNA

<213> Artificial sequence

<220>

<223>

<220>

<221> misc_feature

<222> （103）

<223> H is A or T or C

<220>

<221> misc_feature

<222> （109，115，176，202）

<223> R is A or G

<220>

<221> misc_feature

<222> （168，203）

<223> M is A or C

<400> 1

ttttcgggtt tattacaggg acagcagaga cccagtttgg aaaggaccag ccaaactact 60

ctggaaaggt gaaggggcag tagtaataca agacaataac gahataaarg tagtrccaag 120

aagaaaagca aagatcatta gggattatgg aaaacagatg gcaggtgmtg attgtrtggc 180

aggtagacag gatgaggatt armacatgg 209

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223>

<220>

<221> misc_feature

<222> （4）

<223> H is A or T or C

<220>

<221> misc_feature

<222> （10，16）

<223> R is A or G

<400> 2

cgahataaar gtagtrccaa gaag 24

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223>

<220>

<221> misc_feature

<222> （7）

<223> K is T or G

<220>

<221> misc_feature

<222> （8）

<223> Y is C or T

<400> 3

ccatgtkyta atcctcatcc t 21

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223>

<220>

<221> misc_feature

<222> （11）

<223> M is A or C

<220>

<221> misc_feature

<222> （19）

<223> R is A or G

<400> 4

atggcaggtg mtgattgtrt ggca 24

<210> 5

<211> 211

<212> DNA

<213> Artificial sequence

<220>

<223>

<220>

<221> misc_feature

<222> （103）

<223> H is A or T or C

<220>

<221> misc_feature

<222> （109，115，204）

<223> R is A or G

<220>

<221> misc_feature

<222> （205）

<223> M is A or C

<400> 5

ttttcgggtt tattacaggg acagcagaga cccagtttgg aaaggaccag ccaaactact 60

ctggaaaggt gaaggggcag tagtaataca agacaataac gahataaarg tagtrccaag 120

aagaaaagca aagatcatta gggattatgg aaaacagtct ccaatacctt atccctcgcc 180

tccggtagac aggatgagga ttarmacatg g 211

<210> 6

<211> 26

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 6

tctccaatac cttatccctc gcctcc 26

Claims

1. The kit for detecting HIV-1 comprises a primer pair HIV-1 and a probe HIV-1; the primer pair HIV-1 consists of a primer HIV-F and a primer HIV-R;

the primer HIV-F is a single-stranded DNA molecule shown in a sequence 2 in a sequence table;

the primer HIV-R is a single-stranded DNA molecule shown in a sequence 3 in a sequence table;

the probe HIV-1 is a single-stranded DNA molecule shown in a sequence 4 in a sequence table;

the probe HIV-1 has a fluorescent label at its end.

2. The kit of claim 1, wherein: the kit further comprises an internal standard and an internal standard detection probe; the internal standard is a single-stranded RNA molecule; the DNA fragment obtained by internal standard reverse transcription is a single-stranded DNA molecule shown in a sequence 5 of a sequence table;

the internal standard detection probe is a single-stranded DNA molecule consisting of 20-30 nucleotides, and is the same as a partial segment in a DNA fragment obtained by internal standard reverse transcription.

3. The kit of claim 2, wherein: the internal standard detection probe is a single-stranded DNA molecule shown in a sequence 6 in a sequence table.

4. The kit of any one of claims 1 to 3, wherein: the kit further comprises a working standard; the working standard is a single-stranded RNA molecule; the DNA fragment obtained by reverse transcription of the working standard is shown as a sequence 1 in a sequence table.

5. A kit comprising a kit of parts according to any one of claims 1 to 4.

6, d1) or d 3):

d1) use of a kit of parts according to any one of claims 1 to 4 for the preparation of a kit for the detection of HIV-1;

d3) use of a kit of parts according to any one of claims 1 to 4 for the manufacture of a kit for the identification of HIV-1.