CN111206117A - Kit for detecting human immunodeficiency virus - Google Patents
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Abstract
The invention discloses a kit for detecting human immunodeficiency virus, and relates to the technical field of nucleic acid detection. The kit disclosed by the invention comprises: primer probe premix, positive quality control substances, negative quality control substances and reaction premix. The kit designs a primer and probe combination with a specific sequence aiming at the human immunodeficiency virus, and has high specificity and good coverage; the hole inlet efficiency of the chip is greatly improved, and the reaction false positive is effectively reduced by using hot start Taq enzyme and UDG enzyme; the kit can effectively detect a sample with the final concentration of DNA of only 1 ng/muL, effectively distinguish a standard substance with the final concentration as low as 1 copy/muL, has higher sensitivity, accuracy and stability, can absolutely quantify, has quicker reaction time and lower cost. The application range of the kit is expanded, the technical requirement of clinical ultra-early screening can be met, and the kit can be widely applied to dynamic monitoring of HIV activity and evaluation of curative effect of antiviral drugs in clinic.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a kit for detecting human immunodeficiency virus.
Background
Human Acquired Immunodeficiency Syndrome (AIDS), also known as AIDS, is an infectious disease caused by Human Immunodeficiency Virus (HIV). In 1983, HIV was first discovered in the United states. It is a kind of lentivirus infecting human immune system cell, belongs to one kind of retrovirus, and is divided into two large sizes of HIV-1 and HIV-2 according to the genotype difference, and each large size includes several subtypes. HIV destroys T lymphocyte of human body, and then blocks cellular immunity and humoral immunity process, leading to immune system paralysis, thus leading to spread of various diseases in human body, and finally leading to AIDS.
At present, the types of methods for detecting HIV are many, and mainly comprise HIV antibody detection, HIV nucleic acid detection and the like. The HIV antibody detection comprises a screening test and a confirmation test, the screening test comprises an ELISA method, a chemiluminescence method or an immunofluorescence test and the like, and the commonly used method for the confirmation test is an immunoblotting method, and the method is simple, rapid and low in cost, but is low in sensitivity and accuracy and incapable of quantifying. Especially in low concentration samples, the screening test for newly infected patients in the window period can also be negative. Common methods for HIV nucleic acid detection are reverse transcription PCR (RT-PCR), branched DNA signal amplification system (bDNA), and real time fluorescent quantitative PCR amplification (real time-PCR). Although the nucleic acid detection method has relatively high sensitivity, the method is very important for monitoring the progress of diseases, observing antiviral curative effects and monitoring drug resistance. Clinical significance of viral load assays includes predicting disease progression, providing evidence for starting antiviral therapy, assessing therapeutic efficacy, guiding therapeutic regimen adjustment, and reference indices for early diagnosis of HIV infection. However, the method needs to rely on standard substances with different concentrations to prepare a standard curve for quantification, so that the detection cost is greatly increased, and the detection result is relative quantification which cannot be realized absolutely. Meanwhile, operators are required to have certain professional qualities, and the requirements on the laboratory environment are high. In addition, for samples with very low viral load, false negative results appear in the detection, and the sensitivity is greatly reduced.
The digital PCR (digital PCR) technique is the third generation PCR technique that has been developed in recent years, and generally requires that a sample is diluted to a single molecule level and is evenly distributed into tens of thousands to millions of units for reaction, and the concentration of nucleic acid target molecules is calculated through poisson distribution according to the relative proportion of amplified fluorescent signals in each reaction unit and the volume of the reaction unit. The digital PCR technology does not need to adopt reference genes and standard curves, has high accuracy and good reproducibility, can realize absolute quantitative analysis, and greatly improves the detection sensitivity and the detection rate. At present, the more mature digital PCR platforms are mainly the micro-drop digital PCR of the berle company and the chip digital PCR of the seemefly company. However, the operation steps of the digital PCR instrument are multiple and complex, and the micro-drop type digital PCR is inaccurate in detection of ultra-low content objects due to relative loss of liquid drops and needs an additional quality control system; the chip type digital PCR also needs a plurality of instrument combinations, is expensive and inconvenient to operate, and cannot meet the requirement of convenient clinical use.
Most of the existing kits for detecting HIV nucleic acid utilize real-time fluorescent quantitative PCR for detection. Chinese patent CN106498095A discloses a human immunodeficiency virus type 2 nucleic acid detection kit, which utilizes real-time fluorescent quantitative PCR to detect samples with various concentration gradients, and the detection sensitivity is 15 IU/mL. The kit and the detection method thereof have the advantages of low sensitivity, complex operation, incapability of realizing absolute quantification and unsuitability for detecting samples with extremely low concentration. Chinese patent CN105018647B discloses a kit for detecting HPV16-18 based on digital PCR accurate quantitative typing and a detection method thereof, wherein the kit is based on a digital PCR platform, absolute quantitative detection is carried out on HPV, a sample of 1 copy/muL can be detected, for a sample with lower concentration, compared with fluorescent quantitative PCR, the detection result of the digital PCR method is more accurate, and the kit has good typing detection effect.
Therefore, the invention provides a kit for detecting human immunodeficiency virus based on chip-based digital PCR. The kit can detect HIV-cDNA in body fluid samples such as blood, serum, semen, vaginal secretion, saliva and the like, and provides absolute quantification. The method is more convenient to operate, can be used for accurately qualitatively and quantitatively detecting the extremely-low detection target object, is higher in sensitivity, is stable and reliable in data result, and greatly reduces the detection cost.
Disclosure of Invention
The invention aims to provide a kit for detecting human immunodeficiency virus, which overcomes the problems in the prior art, can accurately perform qualitative and quantitative detection on an extremely low detection target object, and improves the sensitivity.
The invention provides a kit for detecting human immunodeficiency virus on one hand, and the kit comprises primer probe premix;
the primer probe premix comprises a primer pair of HIV-cDNA, a primer pair of human GAPDH gene, a fluorescent probe of HIV-cDNA and a fluorescent probe of human GAPDH gene;
the primer pair of the HIV-cDNA is as follows:
an upstream primer: 5'-TTGACTAGCGGAGGCTAGA-3', as shown in SEQ ID NO: 1;
a downstream primer: 5'-CCCTGGCCTTAACCGAAT-3', as shown in SEQ ID NO: 2;
the primer pair of the human GAPDH gene is as follows:
an upstream primer: 5'-CTAGCTGGCCCGATTTCTCC-3', as shown in SEQ ID NO. 3;
a downstream primer: 5'-CGCCCAATACGACCAAATCAGA-3', as shown in SEQ ID NO. 4;
the nucleotide sequence of the fluorescent probe of the HIV-cDNA is as follows:
5 '-CTGACGCTC + TCGCACCCATCTCTC-3', as shown in SEQ ID NO. 5;
the nucleotide sequence of the fluorescent probe of the human GAPDH gene is as follows:
5 '-TCCGGGTGA + TGCTTTTCCTAGA-3' as shown in SEQ ID NO 6;
wherein "+" indicates that the base at the 3' end is a locked nucleic acid modified base.
The structural formula of the locked nucleic acid is shown as the formula (1):
preferably, the 5 'end of the fluorescent probe of the HIV-cDNA and/or human GAPDH gene contains a fluorescence reporter group FAM and/or CY3, and the 3' end contains a fluorescence quencher group BHQ-1 and/or BHQ-2.
Further preferably, the 5 'end of the fluorescent probe of the HIV-cDNA contains a fluorescent reporter group FAM, and the 3' end of the fluorescent probe of the HIV-cDNA contains a fluorescent quencher group BHQ-1; the 5 'end of the fluorescent probe of the human GAPDH gene contains a fluorescent reporter group CY3, and the 3' end of the fluorescent probe contains a fluorescent quenching group BHQ-2.
Preferably, the concentration ratio of the primer pair for detecting HIV-cDNA, the primer pair for detecting human GAPDH gene, the fluorescent probe for HIV-cDNA and the fluorescent probe for detecting human GAPDH gene in the primer probe premix is 2:2:1: 1.
Preferably, the kit also comprises a positive quality control substance and a negative quality control substance;
the positive quality control product is formed by mixing a plasmid containing 20000 copies/mu L HIV-cDNA fragment and equal volume of 10 ng/mu L human normal T lymphocyte cell line DNA; the negative quality control substance is 10 ng/. mu.L human normal T lymphocyte cell line DNA.
Specifically, the human normal T lymphocyte cell line is H9 cell line.
Preferably, the kit further comprises a reaction premix, and the reaction premix comprises but is not limited to: dATP, dCTP, dGTP, dUTP, BSA (bovine serum albumin), hot start Taq enzyme, uracil-DNA-glycosylase (UDG enzyme), ROX, TritonX-100 and thermostable pyrophosphatase;
further preferably, the concentration ratio of the hot start Taq enzyme to the uracil-DNA-glycosylase is 0.5-1.5: 1. More preferably, the concentration ratio of hot start Taq enzyme to uracil-DNA-glycosylase is 1: 1.
Further preferably, the reaction premix comprises 0.4mM of dATP, dCTP, dGTP and dUTP, 10. mu.g/mu.L of BSA, 100nM of ROX, 0.2% of TritonX-100, and 0.2U of thermostable pyrophosphatase.
The invention also provides application of the kit in detecting HIV-cDNA on a chip-type digital PCR platform.
In another aspect of the present invention, there is provided a method for detecting human immunodeficiency virus using the above kit, wherein the method comprises the following steps:
(1) preparing a digital PCR reaction system: mixing a DNA template to be detected with the primer probe premix and the reaction premix in the kit to obtain a digital PCR reaction system;
the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix in the PCR reaction system is 1:2: 4-6; preferably, the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix is 1:2: 5;
(2) manufacturing a PCR micro-reaction unit according to the digital PCR reaction system obtained in the step (1), and carrying out PCR amplification reaction to obtain an amplification product;
the amplification conditions were: digesting with UDG at 37 deg.C for 300s, hot starting at 95 deg.C for 10min, and circulating for 1 cycle; denaturation at 95 ℃ for 15s, extension at 60 ℃ for 45s, and 40 cycles in total; terminating the reaction at 10 ℃;
(3) and (3) collecting fluorescence signals of the amplification products obtained in the step (2), and judging whether the sample to be detected contains the DNA template of the human immunodeficiency virus and the quantity and the content of the DNA template according to the type of the fluorescence signals.
The DNA template to be detected is derived from body fluid samples such as blood, serum, semen, vaginal secretion or saliva and the like.
Specifically, the method for detecting the human immunodeficiency virus by using the kit comprises the following steps:
(1) preparing a digital PCR reaction system: mixing a DNA template to be detected with the primer probe premix and the reaction premix in the kit to obtain a digital PCR reaction system;
a target fragment containing HIV-cDNA of 500bp in length was synthesized and ligated into a vector plasmid. Transferring into Escherichia coli screening positive bacteria for cloning, extracting purified plasmid, and cutting plasmid into linear by restriction endonuclease; culturing a human normal T lymphocyte cell line, extracting DNA of the human normal T lymphocyte cell line, quantifying by using the Qubit 3.0, and then diluting to the concentration of 10 ng/. mu.L to be used as a background; mixing the plasmid containing HIV-cDNA target segment with human normal T lymphocyte cell line DNA to prepare the DNA template to be detected. Double-stranded DNA quantification was performed with a Qubit and the true copy number was calculated according to the following formula:
(6.02×1023)×(ng/μL×10-9) (DNA length × 660) ═ copies/μ L, where the DNA concentration is in ng/μ L, the DNA length is in bp, and the copy number concentration is in copies/μ L.
The volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix in the PCR reaction system is 0.5-2:2: 5;
(2) manufacturing a PCR micro-reaction unit according to the digital PCR reaction system obtained in the step (1), and carrying out PCR amplification reaction to obtain an amplification product;
the amplification conditions were: digesting with UDG at 37 deg.C for 300s, hot starting at 95 deg.C for 10min, and circulating for 1 cycle; denaturation at 95 ℃ for 15s, extension at 60 ℃ for 45s, and 40 cycles in total; terminating the reaction at 10 ℃;
(3) and (3) collecting fluorescence signals of the amplification products obtained in the step (2), and judging whether the sample to be detected contains the DNA template of the human immunodeficiency virus and the quantity and the content of the DNA template according to the type of the fluorescence signals.
Compared with the prior art, the invention has the advantages that:
(1) the kit for detecting the human immunodeficiency virus is established based on a chip type digital PCR system. The kit designs a primer and probe combination with a specific sequence aiming at the human immunodeficiency virus, and has high specificity and good coverage; by using the autonomously optimized reaction system, the hole-entering efficiency of the chip is greatly improved on the premise of ensuring high efficiency and accuracy of the reaction, the hole-entering rate reaches more than 95%, and the false positive reaction is effectively reduced by using the hot start Taq enzyme and the UDG enzyme.
(2) The kit provided by the invention can effectively detect a sample with the final concentration of DNA of only 1 ng/muL, can effectively distinguish a standard substance with the final concentration as low as 1 copy/muL, has higher sensitivity, accuracy and stability, does not need to prepare a standard curve, can absolutely quantify, has quicker reaction time, lower requirement on a laboratory and lower cost. In addition, the detectable samples have various sources, including blood, serum, semen, vaginal secretion or saliva and other body fluid samples, the application range of the kit, the reaction system and the method is expanded, the technical requirements of clinical ultra-early screening can be met, and the kit can be widely applied to the dynamic monitoring of HIV activity and the evaluation of the curative effect of antiviral drugs.
Detailed Description
The following description of the embodiments is only intended to aid in the understanding of the method of the invention and its core ideas. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The following description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the digital PCR instrument used in this invention is the nucleic acid amplification instrument of patent CN 201911061352.7; the upstream primer, the downstream primer and the nucleotide sequence in the invention are shown in a sequence table in detail.
The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions.
Example 1: preparation of primer probe premix for detecting human immunodeficiency virus
Downloading human immunodeficiency virus gene sequences from an NCBI website, carrying out homologous comparison by utilizing Clustal software, selecting highly conserved regions to design and synthesize a primer pair of HIV-cDNA, a primer pair of human GAPDH gene, a fluorescent probe of HIV-cDNA and a fluorescent probe of human GAPDH gene, wherein the specific sequences are as follows:
the primer pair of HIV-cDNA is:
an upstream primer: 5'-TTGACTAGCGGAGGCTAGA-3', respectively;
a downstream primer: 5'-CCCTGGCCTTAACCGAAT-3', respectively;
primer pairs for the human GAPDH gene were:
an upstream primer: 5'-CTAGCTGGCCCGATTTCTCC-3', respectively;
a downstream primer: 5'-CGCCCAATACGACCAAATCAGA-3', respectively;
the nucleotide sequence of the fluorescent probe of the HIV-cDNA is as follows:
5 '-CTGACGCTC + TCGCACCCATCTCTC-3' as shown in SEQ ID NO: 5;
the nucleotide sequence of the fluorescent probe of the human GAPDH gene is as follows:
5 '-TCCGGGTGA + TGCTTTTCCTAGA-3' as shown in SEQ ID NO 6;
wherein "+" indicates that the base at the 3' end is a locked nucleic acid modified base.
Both primers and probes were synthesized by Shanghai Biotech and purified using HPLC grade. The 5 'end of the fluorescent probe of the HIV-cDNA contains a fluorescent reporter group FAM, and the 3' end of the fluorescent probe contains a fluorescent quenching group BHQ-1; the 5 'end of the fluorescent probe of the human GAPDH gene contains a fluorescent reporter group CY3, and the 3' end of the fluorescent probe contains a fluorescent quenching group BHQ-2. The fluorescent reporter group and the quencher group can be reasonably selected according to a specific platform.
The primer pair of synthesized HIV-cDNA, the primer pair of human GAPDH gene, the fluorescent probe of HIV-cDNA and the fluorescent probe of human GAPDH gene are mixed according to the concentration ratio of 2:2:1:1 to prepare primer probe premix. Namely: the primer probe premix comprises HIV-cDNA upstream and downstream primers, and human GAPDH gene upstream and downstream primer concentrations are both 2 mu M; the concentration of the HIV-cDNA fluorescent probe and the concentration of the human GAPDH gene fluorescent probe are both 1 mu M.
Example 2: kit for detecting human immunodeficiency virus
The kit provided by the invention comprises: the primer probe premix, the positive quality control material, the negative quality control material and the reaction premix provided in example 1.
Preparing a positive quality control product: (1) artificially synthesizing an HIV-cDNA target fragment with the length of 500bp, connecting and loading the target fragment into a vector plasmid pUC57 plasmid (Promega), transferring into escherichia coli screening positive bacteria for cloning, extracting and purifying the plasmid, cutting the plasmid into linearity by using restriction endonuclease, and preparing a plasmid containing 20000 copies/mu L HIV-cDNA fragments; (2) culturing a human normal T lymphocyte cell line, extracting DNA of the human normal T lymphocyte cell line, quantifying by using the Qubit 3.0, and then diluting to the concentration of 10 ng/. mu.L to be used as a background; (3) plasmid containing 20000 copies/. mu.L HIV-cDNA fragment is mixed with equal volume of 10 ng/. mu.L human normal T lymphocyte cell line DNA to prepare mixed solution containing 10000 copies/. mu.L HIV-cDNA, which is positive quality control product.
Negative quality control product: culturing a human normal T lymphocyte cell line, extracting DNA of the human normal T lymphocyte cell line, quantifying by using the Qubit 3.0, and then diluting to the concentration of 10 ng/mu L, thus obtaining the negative quality control product.
The reaction premix comprises: concentrations of dATP, dCTP, dGTP and dUTP were all 0.4mM, concentration of BSA was 10. mu.g/. mu.L, concentration of ROX was 100nM, concentration percentage of TritonX-100 was 0.2%, concentration of thermostable pyrophosphatase was 0.2U, concentration of hot start Taq enzyme was 1U, concentration of UDG enzyme was 1U, i.e., concentration ratio of hot start Taq enzyme to UDG enzyme was 1: 1.
In 20U LPCR reaction system, the final concentrations of dATP, dCTP, dGTP and dUTP were all 0.2mM, the final concentration of BSA was 5. mu.g/. mu.L, the final concentration of ROX was 50nM, the final concentration of TritonX-100 was 0.1%, and the final concentration of thermostable pyrophosphatase was 0.1U.
Example 3: method for detecting human immunodeficiency virus
(1) Preparing a digital PCR reaction system: the DNA template to be tested is mixed with the DNA template in the kit described in example 2Mixing the primer probe premix with the reaction premix according to ddH2Adding the samples into a PCR tube according to the reaction system in the table 1 in the sequence of O, reaction premix, primer probe premix and DNA template, uniformly mixing the mixed system for 15s by using soft vortex, and collecting the solution to the bottom of the test tube by short-time centrifugation to obtain a digital PCR reaction system; the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix in the reaction system is 1:2: 5.
Table 1: reaction system
(2) And (2) loading the digital PCR reaction system obtained in the step (1) onto a PCR chip to form a micro-reaction unit. The chip is placed into a digital PCR instrument, and PCR reaction is carried out according to the PCR reaction conditions in the table 2, so as to obtain an amplification product.
Table 2: PCR reaction conditions
(3) And (3) collecting a fluorescence signal of the amplification product obtained in the step (2), and selecting FAM and CY3 as channels for fluorescence detection. And judging the effective fluorescence positive points of the two channels by computer analysis, analyzing the result, and judging whether the sample to be detected contains the DNA template of the human immunodeficiency virus and the quantity and the content of the DNA template according to the type of the fluorescence signal.
Example 4: detection of standards
Preparation of HIV-cDNA-containing standard: (1) artificially synthesizing target fragment containing HIV-cDNA with length of 500bp, connecting and loading it into plasmid pUC57 (Promega), transferring it into colibacillus screening positive bacteria, cloning, extracting purified plasmid, cutting plasmid into linear by restriction endonuclease, preparing DNA fragment containing copy number concentration of 2X 101-7Plasmid solution of HIV-cDNA target fragment of copies/mu L; (2) human normal T lymphocyte cell line was cultured, DNA was extracted, quantified using Qubit 3.0, and then diluted to 10 ng/. mu.LConcentration as background; (3) 2X 101-7Plasmid containing the target fragment of copies/. mu.L HIV-cDNA was mixed with an equal volume of 10 ng/. mu.L human normal T lymphocyte cell line DNA to prepare a plasmid containing 1X 101-7Standards of copies/. mu.L HIV-cDNA, numbered 1-7.
The HIV-cDNA standards of 7 different concentrations obtained by the above method, negative quality control (control group) in the kit of example 2 and no template sample control (NTC) were tested by the method of example 3, and the experiment was repeated 3 times. The results are shown in Table 3.
Table 3: test results of the standards
As can be seen from Table 3, the kit provided by the invention can effectively detect a sample with the final concentration of DNA of 1 ng/muL, can effectively distinguish a standard substance with the final concentration of 1 copy/muL, and has the advantages of accurate detection, high stability, 98.6% of pore-entering rate and no false positive reaction.
Example 5: kit for detecting human immunodeficiency virus and detection method thereof
A kit for detecting human immunodeficiency virus was distinguished from the kit described in example 2 only in that the concentration of hot-start Taq enzyme and the concentration of UDG enzyme in the reaction premix were 0.5U and 1U, i.e., the ratio of the concentrations of hot-start Taq enzyme and UDG enzyme was 0.5: 1.
The kit is used for detecting the standard substance in the embodiment 4, and the detection method is different from the method in the embodiment 3 only in that the digital PCR reaction system is prepared in the step (1), wherein the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix is 1:2:4, namely: in a 20-microliter reaction system, 2 microliter of DNA template to be detected, 4 microliter of primer probe premix, 8 microliter of reaction premix and ddH2O6. mu.L. The results are shown in Table 4.
Table 4: test results of the standards
The results show that: the kit and the detection method provided by the invention can effectively detect a sample with the final concentration of DNA of only 1 ng/muL, can effectively distinguish a standard substance with the final concentration of 1 copy/muL, have the hole entering rate of 96.7 percent and have no false positive reaction.
Example 6: kit for detecting human immunodeficiency virus and detection method thereof
A kit for detecting human immunodeficiency virus was distinguished from the kit described in example 2 only in that the concentration of hot-start Taq enzyme and the concentration of UDG enzyme in the reaction premix were 1.5U and 1U, i.e., the concentration ratio of hot-start Taq enzyme to UDG enzyme was 1.5: 1.
The kit is used for detecting the standard substance in the embodiment 4, and the detection method is different from the method in the embodiment 3 only in that the digital PCR reaction system is prepared in the step (1), wherein the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix is 1:2:6, namely: in a 20-microliter reaction system, 2 microliter of DNA template to be detected, 4 microliter of primer probe premix, 12 microliter of reaction premix and ddH2O2. mu.L. The results are shown in Table 5.
Table 5: test results of the standards
The results show that: the kit and the detection method provided by the invention can effectively detect a sample with the final concentration of DNA of only 1 ng/muL, can effectively distinguish a standard substance with the final concentration of 1 copy/muL, have the hole entering rate of 98.4 percent and have no false positive reaction.
Example 7: kit for detecting human immunodeficiency virus and detection method thereof
A kit for detecting human immunodeficiency virus was distinguished from the kit described in example 2 only in that the concentration of hot-start Taq enzyme and the concentration of UDG enzyme in the reaction premix were 0.5U and 1U, i.e., the ratio of the concentrations of hot-start Taq enzyme and UDG enzyme was 0.5: 1.
The kit is used for detecting the standard substance in the embodiment 4, and the detection method is different from the method in the embodiment 3 only in that the digital PCR reaction system is prepared in the step (1), wherein the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix is 1:2:6, namely: in a 20-microliter reaction system, 2 microliter of DNA template to be detected, 4 microliter of primer probe premix, 12 microliter of reaction premix and ddH2O2. mu.L. The results are shown in Table 6.
Table 6: test results of the standards
The results show that: the kit and the detection method provided by the invention can effectively detect a sample with the final concentration of DNA of only 1 ng/muL, can effectively distinguish a standard substance with the final concentration of 1 copy/muL, have the hole entering rate of 97.3 percent and have no false positive reaction.
Example 8: kit for detecting human immunodeficiency virus and detection method thereof
A kit for detecting human immunodeficiency virus was distinguished from the kit described in example 2 only in that the concentration of hot-start Taq enzyme and the concentration of UDG enzyme in the reaction premix were 1.5U and 1U, i.e., the concentration ratio of hot-start Taq enzyme to UDG enzyme was 1.5: 1.
The kit is used for detecting the standard substance in the embodiment 4, and the detection method is different from the method in the embodiment 3 only in that the digital PCR reaction system is prepared in the step (1), wherein the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix is 1:2:4, namely: in a 20-microliter reaction system, 2 microliter of DNA template to be detected, 4 microliter of primer probe premix, 8 microliter of reaction premix and ddH2O6. mu.L. The results are shown in Table 7.
Table 7: test results of the standards
The results show that: the kit and the detection method provided by the invention can effectively detect a sample with the final concentration of DNA of only 1 ng/muL, can effectively distinguish a standard substance with the final concentration of 1 copy/muL, have the hole entering rate of 97.9 percent and have no false positive reaction.
Example 9: sample detection for aids patients
Blood samples were tested using the kit of example 2 and the method of example 3, and the samples were obtained from 20 clinically confirmed aids patients in a sample volume of 5 mL. When The sample is detected by using The method, an immunoblotting reagent, namely HIV-Blot detection kit, which is produced by Genelabs company is used as a reference, The AMPLICOR HIV 1 Test (v1.5) reagent, which is produced by Roche (ROCHE) company, is used for rechecking, The sample is judged to be true negative (positive) when The detection result is consistent with The rechecking result, and The sample is judged to be false negative (positive) when The detection result is inconsistent with The rechecking result, and then The sample is compared with a clinical diagnosis result.
The method for extracting RNA in the sample comprises the following steps: 1mL of blood sample was aspirated from a blood collection tube, and RNA in the sample was extracted and purified using QIAamp Viral RNAMINi Kit (purchased from QIAGEN, Cat number/ID: 52904), followed by SuperScriptTMIII Reverse transcription was performed using the Reverse transcription Transcriptase kit (purchased from Invitrogen, cat # 18080044). The total amount of the quantit is confirmed to be not less than 20ng after the quantit is quantified, the kit in the embodiment 2 is adopted to carry out detection according to the method in the embodiment 3, and the detection results are shown in a table 8.
Table 8: test results of clinical specimens
The detection result shows that: after rechecking, the kit provided by the invention detects 20 positive cases in the result, and the false positive rate is 5%; the results of HIV-Blot detection kits produced by Genelabs company contain 15 positive cases, the false positive rate is 0, and the false negative rate is 10%; the accuracy of the kit provided by the invention is 95%, and the accuracy of the HIV-Blot detection kit produced by Genelabs company is 90%.
Clinical diagnosis results show that 20 AIDS patient samples are positive, which indicates that the accuracy of the kit provided by the invention is 100%, and the sensitivity is higher.
The result shows that the kit provided by the invention can meet the technical requirement of clinical ultra-early screening, and can be widely applied to the dynamic monitoring of HIV activity and the evaluation of the curative effect of antiviral drugs. Compared with the existing detection kit, the detection kit has the advantages of more convenient operation, stable and reliable data result and greatly reduced detection cost.
The inventor of the application also detects samples from body fluids such as blood, serum, semen, vaginal secretion or saliva, and the kit provided by the invention can effectively detect DNA samples with extremely low concentration, can effectively distinguish standard substances with the final concentration of 1 copy/mu L, has the pore-entering rate of more than 95 percent, and has no false positive reaction.
Comparative example 1
The kit provided in this comparative example is different from the kit described in example 2 only in that the concentration of hot start Taq enzyme and the concentration of UDG enzyme in the reaction premix were 0.1U and 1U, respectively. The kit was used to detect the standard described in example 4, and the detection method was the method described in example 3. The results are shown in Table 9.
Table 9: the result of the detection
Note: "-" indicates no detection.
The detection result shows that: the kit provided by the invention can effectively detect a sample with the final concentration of DNA of only 1 ng/mu L, can effectively distinguish a standard substance with the final concentration of 100 copies/mu L, has the hole inlet rate of 97.4 percent, and has high reaction false negative rate for a sample with lower concentration.
Comparative example 2
Using the kit of example 2Detecting the standard substance in the embodiment 4, wherein the detection method is different from the method in the embodiment 3 only in that the digital PCR reaction system is prepared in the step (1), and the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix is 1:2:3.5, namely: in a 20-microliter reaction system, 2 microliter of DNA template to be detected, 4 microliter of primer probe premix, 7 microliter of reaction premix and ddH2O7. mu.L. The results are shown in Table 10.
Table 10: the result of the detection
Note: "-" indicates no detection.
The detection result shows that: the kit provided by the invention can detect a sample with the final concentration of DNA of only 1 ng/mu L, can effectively distinguish a standard substance with the final concentration of 10 copies/mu L, and has the advantages of 95.8% of hole entering rate, high false negative rate of reaction and lower accuracy.
Comparative example 3
The kit used in this comparative example differs from the kit described in example 2 only in that the fluorescent probe used is not modified by a locked nucleic acid. The kit was used to detect the standard described in example 4, and the detection method was the method described in example 3. The results are shown in Table 11.
Table 11: the result of the detection
Note: "-" indicates no detection.
The detection result shows that: the kit provided by the invention can detect a sample with the final concentration of DNA of only 1 ng/mu L, can effectively distinguish a standard substance with the final concentration of 10 copies/mu L, has a hole entering rate of 96.7 percent, a high false negative rate of reaction and low accuracy.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
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<120> a kit for detecting human immunodeficiency virus
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Claims (10)
1. The kit for detecting the human immunodeficiency virus is characterized by comprising primer-probe premix;
the primer probe premix comprises a primer pair of HIV-cDNA, a primer pair of human GAPDH gene, a fluorescent probe of HIV-cDNA and a fluorescent probe of human GAPDH gene;
the primer pair of the HIV-cDNA is as follows:
an upstream primer: 5'-TTGACTAGCGGAGGCTAGA-3', respectively;
a downstream primer: 5'-CCCTGGCCTTAACCGAAT-3', respectively;
the primer pair of the human GAPDH gene is as follows:
an upstream primer: 5'-CTAGCTGGCCCGATTTCTCC-3', respectively;
a downstream primer: 5'-CGCCCAATACGACCAAATCAGA-3', respectively;
the nucleotide sequence of the fluorescent probe of the HIV-cDNA is as follows:
5’-CTGACGCTC+TCGCACCCATCTCTC-3’；
the nucleotide sequence of the fluorescent probe of the human GAPDH gene is as follows:
5’-TCCGGGTGA+TGCTTTTCCTAGA-3’；
wherein "+" indicates that the base at the 3' end is a locked nucleic acid modified base.
2. The kit according to claim 1, wherein the fluorescent probe for HIV-cDNA and/or human GAPDH gene comprises a fluorescent reporter group FAM and/or CY3 at the 5 'end and a fluorescent quencher group BHQ-1 and/or BHQ-2 at the 3' end.
3. The kit according to claim 1, wherein the fluorescent probe of HIV-cDNA has a fluorescent reporter group FAM at the 5 'end and a fluorescent quencher group BHQ-1 at the 3' end;
the 5 'end of the fluorescent probe of the human GAPDH gene contains a fluorescent reporter group CY3, and the 3' end of the fluorescent probe contains a fluorescent quenching group BHQ-2.
4. The kit according to claim 1, wherein the concentration ratio of the primer pair for detecting HIV-cDNA, the primer pair for detecting human GAPDH gene, the fluorescent probe for HIV-cDNA and the fluorescent probe for detecting human GAPDH gene in the primer probe premix is 2:2:1: 1.
5. The kit of claim 1, wherein the kit further comprises a positive quality control material and a negative quality control material;
the positive quality control product is formed by mixing a plasmid containing 20000 copies/mu L HIV-cDNA fragment and equal volume of 10 ng/mu L human normal T lymphocyte cell line DNA; the negative quality control substance is 10 ng/. mu.L human normal T lymphocyte cell line DNA.
6. The kit of claim 1, further comprising a reaction premix: dATP, dCTP, dGTP, dUTP, BSA, hot start Taq enzyme, uracil-DNA-glycosylase, ROX, TritonX-100 and thermostable pyrophosphatase.
7. The kit of claim 6, wherein the reaction premix contains 0.4mM of dATP, dCTP, dGTP and dUTP, 10. mu.g/μ L of BSA, 100nM of ROX, 0.2% of TritonX-100 and 0.2U of thermostable pyrophosphatase.
8. The kit according to claim 6, wherein the concentration ratio of the hot start Taq enzyme to the uracil-DNA-glycosylase is 0.5-1.5: 1.
9. Use of the kit according to any one of claims 1 to 8 for the detection of HIV-cDNA on a chip-based digital PCR platform.
10. A method for detecting human immunodeficiency virus using the kit of any one of claims 1 to 8, comprising the steps of:
(1) preparing a digital PCR reaction system: mixing a DNA template to be detected with the primer probe premix solution and the reaction premix solution in the kit of any one of claims 1 to 8 to obtain a digital PCR reaction system;
the volume ratio of the DNA template to be detected, the primer probe premix and the reaction premix in the PCR reaction system is 1:2: 4-6;
(2) manufacturing a PCR micro-reaction unit according to the digital PCR reaction system obtained in the step (1), and carrying out PCR amplification reaction to obtain an amplification product;
the amplification conditions were: digesting with UDG at 37 deg.C for 300s, hot starting at 95 deg.C for 10min, and circulating for 1 cycle; denaturation at 95 ℃ for 15s, extension at 60 ℃ for 45s, and 40 cycles in total; terminating the reaction at 10 ℃;
(3) and (3) collecting fluorescence signals of the amplification products obtained in the step (2), and judging whether the sample to be detected contains the DNA template of the human immunodeficiency virus and the quantity and the content of the DNA template according to the type of the fluorescence signals.
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| CN112210625A (en) * | 2020-11-02 | 2021-01-12 | 中山大学达安基因股份有限公司 | Kit and method for quantitatively detecting human immunodeficiency virus type 1DNA (deoxyribonucleic acid) load |
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Application publication date: 20200529 |