Primer and kit for rapidly detecting HIV-II virus based on fluorescent quantitative PCR and application

Abstract

The invention provides a primer and a kit for rapidly detecting an HIV-II virus based on fluorescent quantitative PCR (Polymerase Chain Reaction) and application, and belongs to the technical field of biological detection. The primer for rapidly detecting the HIV-II virus based on the fluorescent quantitative PCR comprises a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2. Whether HIV-II virus pollution exists in stem cells or not can be detected in real time by detecting the HIV-II virus based on the primer, and the problem of false negative is avoided. Meanwhile, the detection method provided by the invention has good detection sensitivity and detection specificity, and provides a new quality control means for stem cell separation and culture.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a primer, a kit and an application for rapid detection of HIV-II virus based on fluorescent quantitative PCR. Background Art

[0002] Stem cell drugs are derived from primary cultures of human tissues such as the placenta and umbilical cord. The collected materials (maternal blood and umbilical cord blood) need to be tested for human viruses before they can enter the subsequent production process. At the same time, based on the relevant provisions of the guidelines for preclinical pharmaceutical research of stem cell drugs, it is necessary to set up appropriate quality control points (such as seed cell banks or working cell banks) to test the stem cells for human viruses. Since the human virus detection performed at the collection stage is a release test based on the colloidal gold / ELISA method, the detection method is not directly for virus detection, but indirectly detects the presence of the virus by detecting viral antibodies in the blood sample. It takes a certain amount of time for the donor to produce antibodies after being infected with the virus. If the virus antibody test is performed during this window period, the result is likely to be a false negative, which poses a certain risk. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide a primer for rapid detection of HIV-II virus based on fluorescent quantitative PCR, which directly detects HIV-II virus, is not affected by false negatives in the window period, and can directly detect stem cell samples, thereby achieving high sensitivity and high specificity detection.

[0004] The invention provides a primer for rapid detection of HIV-II virus based on fluorescent quantitative PCR, comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2.

[0005] The invention provides a kit for rapidly detecting HIV-II virus based on fluorescent quantitative PCR, comprising the primers.

[0006] Preferably, a qPCR detection premix and a positive control are also included.

[0007] Preferably, the qPCR detection premix includes 2×TSINGKE Master qPCR Mix-SYBR (+UDG).

[0008] Preferably, the positive control comprises a recombinant plasmid containing a DNA fragment of HIV-II virus.

[0009] The invention provides application of the primer in preparing a kit for detecting HIV-II virus infection.

[0010] The invention provides application of the primer in preparing a kit for HIV-II virus contamination in the process of stem cell separation or culture.

[0011] The present invention provides a method for rapidly detecting HIV-II virus contamination in stem cell separation or culture based on fluorescent quantitative PCR, comprising the following steps:

[0012] Extracting total RNA from the sample to be tested, and performing reverse transcription to obtain cDNA of the sample to be tested;

[0013] Using the cDNA of the sample to be tested as a template, the primers are used for qPCR amplification, and judging whether there is HIV-II virus contamination in the sample to be tested according to the Ct value of the test result:

[0014] A positive result is when the CT value is ≤35.0 and the curve has an obvious exponential growth curve, indicating that the sample to be tested is contaminated with HIV-II virus;

[0015] A test result of CT>40 or no Ct value and no obvious amplification curve is considered a negative result, indicating that the sample to be tested is not contaminated by HIV-II virus.

[0016] Preferably, the reaction system for qPCR amplification is 10 μl, comprising the following components: 5 μl of 2×TSINGKE MasterqPCR Mix-SYBR (+UDG), 0.5 μl of 10 μM upstream primer, 0.5 μl of 10 μM downstream primer and 4 μl of sample RNA.

[0017] Preferably, the reaction procedure of the qPCR amplification is pre-denaturation at 50°C for 2 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 40 cycles, and collecting fluorescence signals at 72°C.

[0018] The present invention provides a primer for rapid detection of HIV-II virus based on fluorescent quantitative PCR, including a forward primer with a nucleotide sequence as shown in SEQ ID NO:1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:2. The present invention designs a pair of specific detection primers for the genome sequence of HIV-II virus based on the real-time fluorescent quantitative PCR method, amplifies by qPCR, and determines whether HIV-II virus exists according to the Ct value of the amplification result. It can be seen that the detection of HIV-II virus based on primers can detect whether HIV-II virus infection exists in the sample to be tested in real time, avoiding the problem of false negative. At the same time, the detection limit of the detection primer provided by the present invention is 5×10 3IU / ml, which has obvious detection advantages compared with commercial kits. In addition, the primers are used for qPCR detection, and the melting curve is a single peak, indicating that the detection reaction has no non-specific amplification and no primer dimer formation, and can only amplify HIV-II virus, with good detection specificity. Therefore, the primers provided by the present invention provide a new detection method for whether stem cells are contaminated with HIV-II virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The melting curve of the primers was used to detect HIV-II virus. DETAILED DESCRIPTION

[0020] The invention provides a primer for rapid detection of HIV-II virus based on fluorescent quantitative PCR, comprising a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 (5'-TAGTCACAGCGTACATCG-3') and a reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2 (5'-ATCTATCAGCATCCTCCA-3').

[0021] The present invention has no particular limitation on the method for obtaining the primers, and any primer obtaining method known in the art, such as artificial synthesis, can be used. In the embodiment of the present invention, the primers can be synthesized by entrusting Beijing Qingke Biotechnology Co., Ltd.

[0022] The invention provides a kit for rapidly detecting HIV-II virus based on fluorescent quantitative PCR, comprising the primers.

[0023] In the present invention, the kit preferably further comprises a qPCR detection premix and a positive control. The qPCR detection premix comprises 2×TSINGKE Master qPCR Mix-SYBR (+UDG). The positive control comprises a recombinant plasmid containing a DNA fragment of HIV-II virus.

[0024] Based on the fact that the primers have good detection specificity and detection sensitivity for HIV-II virus detection, the present invention provides the use of the primers in preparing a kit for detecting HIV-II virus infection.

[0025] The invention provides application of the primer in preparing a kit for HIV-II virus contamination in the process of stem cell separation or culture.

[0026] The present invention has no particular limitation on the type of stem cells, and any type of stem cells known in the art, such as mesenchymal stem cells, may be used.

[0027] The present invention provides a method for rapidly detecting HIV-II virus contamination in stem cell separation or culture based on fluorescent quantitative PCR, comprising the following steps:

[0028] Extracting total RNA from the sample to be tested, and performing reverse transcription to obtain cDNA of the sample to be tested;

[0029] Using the cDNA of the sample to be tested as a template, the primers are used for qPCR amplification, and judging whether there is HIV-II virus contamination in the sample to be tested according to the Ct value of the test result:

[0030] A positive result is when the CT value is ≤35.0 and the curve has an obvious exponential growth curve, indicating that the sample to be tested is contaminated with HIV-II virus;

[0031] A negative result is a test result of CT>40 or no Ct value and no obvious amplification curve, indicating that the sample to be tested is not contaminated with HIV-II virus; if the test result CT value is>35.0 and ≤40, repeat the test.

[0032] The present invention extracts total RNA of the sample to be tested, and obtains cDNA of the sample to be tested through reverse transcription.

[0033] The present invention has no particular limitation on the type of the sample to be tested, and any sample to be tested known in the art can be used, such as umbilical cord tissue, placenta, amniotic membrane, amniotic fluid, isolated stem cells, cultured stem cells, etc. The present invention has no particular limitation on the type of stem cells, and any type of stem cells known in the art can be used, such as mesenchymal stem cells.

[0034] The present invention has no particular limitation on the extraction of total RNA from the sample to be tested, and a cell RNA extraction method known in the art can be used. In an embodiment of the present invention, a blood / cell / tissue genomic RNA extraction kit is used. The present invention has no particular limitation on the reverse transcription, and a reverse transcription method known in the art can be used.

[0035] After obtaining the cDNA of the sample to be tested, the present invention uses the cDNA of the sample to be tested as a template and the primers to perform qPCR amplification, and determines whether HIV-II virus contamination exists in the sample to be tested according to the Ct value of the detection result.

[0036] In the present invention, the reaction system of the qPCR amplification is preferably 10 μl, preferably including the following components: 2×TSINGKE Master qPCR Mix-SYBR (+UDG) 5 μl, 10 μM upstream primer 0.5 μl, 10 μM downstream primer 0.5 μl and sample RNA 4 μl. The reaction procedure of the qPCR amplification is preferably 50°C pre-denaturation for 2 min; 95°C pre-denaturation for 2 min; 95°C denaturation for 15 s, 55°C annealing for 30 s, 72°C extension for 30 s, 40 cycles, and 72°C fluorescence signal collection. Real-time fluorescence quantitative PCR is a real-time detection of the PCR process through fluorescence signals during the PCR amplification process. Since there is a linear relationship between the Ct value of the template and the starting copy number of the template during the exponential period of PCR amplification, it becomes the basis for quantification. The single peak diagram of the PCR product melting curve diagram indicates the singleness of the PCR amplification product. The results show that the melting curve obtained by the primers provided by the present invention for detecting the HIV-II virus positive control (a plasmid containing the HIV-II target fragment) is a single peak, indicating that the primers can only detect the HIV-II virus, but do not produce a single peak for the RNA of mesenchymal stem cells, indicating that the primers are suitable for the detection of cell samples contaminated with HIV-II virus.

[0037] In one embodiment of the present invention, the detection sensitivity experiment of the method was carried out, and the positive control substance (a plasmid containing the target fragment of HIV-II) from the commercial HIV-II detection kit was diluted 10 times to obtain 1×10 6 IU / ml, 1×10 5 IU / ml, 1×10 4 IU / ml, 5×10 3 IU / ml, 5×10 2 IU / ml, the detection limit of the primers for HIV-II virus was 5×10 3 IU / ml, with good detection sensitivity.

[0038] In another embodiment of the present invention, the method was carried out to detect samples simultaneously infected with HIV-II and other types of viruses (HBV). The results showed that infection with other viruses had no effect on the detection of HIV-II virus, which indicates that the method of the present invention is suitable for the detection of samples simultaneously infected with multiple viruses and has good promotion and application value.

[0039] The primers, kit and application for rapid detection of HIV-II virus based on fluorescent quantitative PCR provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] The experimental instruments and reagents are shown in Table 1.

[0041] Table 1 Main instruments

[0042]

[0043]

[0044] Table 2 Main reagents

[0045]

[0046] Example 1

[0047] A kit for rapid detection of HIV-II virus by qPCR

[0048] 1. Design of primers for HIV-II virus detection

[0049] First, find the complete RNA genome sequence of HIV-II virus in the NCBI database (GI number: 9628880). Use the primer design software Premier 5 to design the following pair of primers, the specific sequences are as follows:

[0050] Upstream primer: 5'-TAGTCACAGCGTACATCG-3' (SEQ ID NO: 1);

[0051] Downstream primer: 5'-ATCTATCAGCATCCTCCA-3' (SEQ ID NO: 2).

[0052] 2. Other components of the kit are shown in Table 3.

[0053] Table 3 Detection reagents in the detection kit

[0054]

[0055]

[0056] Example 2

[0057] A method for rapid detection of HIV-II virus by qPCR

[0058] 1. RNA extraction of the sample group to be tested

[0059] Take 1 ml of the supernatant of the mesenchymal stem cell preparation, centrifuge at 1500 rpm for 5 min to remove the supernatant, and retain the bottom precipitate. Extract genomic RNA according to the instructions of the Tiangen genomic RNA extraction kit to obtain the total RNA of mesenchymal stem cells. At the same time, the positive control in the HIV-II virus commercial kit was used as a positive control, and water was used as a negative control, and HIV-II virus detection was carried out at the same time.

[0060] 2. RNA Reverse Transcription

[0061] a) Take out gDNA remover, 10x gDNA remover Buffer, and RNase-free water from the reverse transcription kit, place them on ice to melt, and centrifuge for a few seconds before use. Take eight qPCR tubes and prepare the reverse transcription reaction system according to Table 4 below:

[0062] Table 4 Reverse transcription reaction system

[0063] Components RNA template (1 μg / μl) gDNA remover 10xgDNA remover Buffer RNase-free water Total volume Dosage 1μl 1μl 1μl Add to 10 μl 10μl

[0064] a) Mix gently with a pipette tip, centrifuge briefly, incubate at 42°C for 2 min, and then incubate at 60°C for 5 min.

[0065] b) The mixture was quickly cooled on ice, centrifuged briefly, and then the components in Table 5 were added:

[0066] Table 5 Reverse transcription reaction components

[0067] Components Usage dNTP Mix 1μl <![CDATA[Goldenstar TM Randomer*]]> 1μl <![CDATA[5xGoldenstar TM Buffer]]> 4μl DTT 1μl <![CDATA[Goldenstar TM RT6]]> 1μl RNase-free water 2μl

[0068] c) Mix gently with a pipette tip, centrifuge briefly, and perform reverse transcription according to the reaction conditions in Table 6.

[0069] Table 6 Reaction conditions

[0070] condition time 25℃ 10min 55℃ 15min

[0071] 3. qPCR Amplification

[0072] The total reaction system was 10 μl, including the following components: 2×TSINGKE Master qPCR Mix-SYBR (+UDG) 5 μl, upstream primer (10 μM) 0.5 μl, downstream primer (10 μM) 0.5 μl, sample RNA 4 μl. The final concentration of upstream and downstream primers (SEQ ID NO: 1 to SEQ ID NO: 2) was 0.5 μM. The same template was used for multiple well reactions, and all reactions were performed on a LightCycler 480 fluorescence quantitative PCR instrument. The reaction procedure of real-time fluorescence quantitative PCR was: 50°C pre-denaturation for 2 min; 95°C pre-denaturation for 2 min; 95°C denaturation for 15 s, 55°C annealing for 30 s, 72°C extension for 30 s, 40 cycles, and fluorescence signal collection at 72°C. Negative control detection wells and positive control detection wells were also set. The negative control was DEPC water. The positive control was the positive control provided in the HIV-II commercial kit.

[0073] 3. Result judgment (qualitative analysis)

[0074] According to the sample group to be tested, the positive control (1×10 6 IU / ml) and the negative control (DEPC water) CT value to determine whether the sample to be tested contains HIV-II virus.

[0075] Positive: The CT value of the test result is ≤35.0, and the curve has an obvious exponential growth curve.

[0076] Negative: test result CT>40 or no Ct value and no obvious amplification curve;

[0077] If the CT value of the test result is >35.0 and ≤40, repeat the test.

[0078] The results are shown in Table 7 and Figure 1 .Depend on Figure 1 It can be seen that the melting curve of the HIV-II virus detection primer is a single peak, indicating that there is no primer dimer and non-specific amplification in the amplification process.

[0079] Table 7 Test results

[0080] Serial number Group CT mean in conclusion 1 Positive Control 23.24 Negative 2 MSC samples / Negative 3 Negative control / Negative

[0081] Example 3

[0082] Detection sensitivity experiment

[0083] 1. The positive control (plasmid containing the HIV-II target fragment) was from a commercial HIV-II detection kit (Shanghai Zhijiang Biotechnology Co., Ltd., catalog number: SR-0021-02), with an initial concentration of 1×10 7 IU / ml. Dilute the positive control with sample diluent to different concentrations as shown in Table 7 below.

[0084] 2. Fluorescence quantitative PCR reaction system and procedure

[0085] The total reaction system was 10 μl, 2×TSINGKE Master qPCR Mix-SYBR (+UDG) 5 μl, upstream primer (10 μM) 0.5 μl, downstream primer (10 μM) 0.5 μl, and sample RNA 4 μl. The final concentration of the upstream and downstream primers was 0.5 μM each. The same template was used for duplicate well reactions, and all reactions were performed on a LightCycler 480 fluorescence quantitative PCR instrument. The reaction procedure for real-time fluorescence quantitative PCR was: 50°C pre-denaturation for 2 min; 95°C pre-denaturation for 2 min; 95°C denaturation for 15 s, 55°C annealing for 30 s, 72°C extension for 30 s, 40 cycles, and fluorescence signal collection at 72°C.

[0086] 3. The experimental results are shown in Table 8.

[0087] Table 8 Test results of positive control substances at different concentrations

[0088] serial number Positive control dilution concentration The kit comes with primers to detect the mean CT value The primers of the present invention detect the mean CT value 1 <![CDATA[1×10 6 IU / ml]]> 28.82 23.85 2 <![CDATA[1×10 5 IU / ml]]> 30.86 25.72 3 <![CDATA[1×10 4 IU / ml]]> 31.96 27.95 4 <![CDATA[5×10 3 IU / ml]]> 33.64 31.67 5 <![CDATA[5×10 2 IU / ml]]> Not detected Not detected

[0089] The results in Table 7 show that the detection limit of the primers of the present invention for HIV-II virus is 5×10 3 IU / ml, and compared with the detection CT mean value of the primers provided by the HIV-II commercial kit, the primers of the present invention have higher sensitivity.

[0090] Example 4

[0091] Specificity Verification I

[0092] 1. The positive control was from a commercial HIV-II test kit with an initial concentration of 1×10 7 IU / ml. In the positive control (1×10 6 IU / ml) was tested by adding half of HBV positive serum.

[0093] 2. Fluorescence quantitative PCR reaction system and procedure

[0094] The reaction system was configured to be 10 μl / sample, including the following components: 2×TSINGKE Master qPCR Mix-SYBR (+UDG) 5 μl, upstream primer (10 μM) 0.5 μl, downstream primer (10 μM) 0.5 μl, sample RNA 4 μl. The final concentration of upstream and downstream primers was 0.5 μM each. The same template was used for duplicate well reactions, and all reactions were performed on a LightCycler 480 fluorescence quantitative PCR instrument. The reaction procedure of real-time fluorescence quantitative PCR was: 50℃ pre-denaturation for 2 min; 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles, and fluorescence signal collection at 72℃.

[0095] 3. The experimental results are shown in Table 9.

[0096] Table 9 Specificity verification results

[0097]

[0098] The results in Table 9 show that the primers provided by the present invention have good specificity and can detect the virus HIV-II in the mixture.

[0099] Example 5

[0100] Exclusiveness Verification II

[0101] 1. The other virus (HAV, HIV-1 and HTLV) reference substances used for specificity verification are all from commercial detection kits, and the working concentration is 1×10 6 IU / ml.

[0102] The kit information is shown in Table 10 below.

[0103] Table 10 Kit information

[0104] Kit Name Part Number Manufacturer HAV Virus Nucleic Acid Assay Kit HR-0001-02 Shanghai River HIV-1 Virus Nucleic Acid Assay Kit SR-0020-02 Shanghai River HTLV Virus Nucleic Acid Assay Kit ZY-15-87800 Zeye Biotechnology

[0105] 2. Fluorescence quantitative PCR reaction system and procedure

[0106] The reaction system was configured to be 10 μl / sample, including the following components: 2×TSINGKE Master qPCR Mix-SYBR (+UDG) 5 μl, upstream primer (10 μM) 0.5 μl, downstream primer (10 μM) 0.5 μl, sample RNA 4 μl. The final concentration of upstream and downstream primers was 0.5 μM each. The same template was used for duplicate well reactions, and all reactions were performed on a LightCycler 480 fluorescence quantitative PCR instrument. The reaction procedure of real-time fluorescence quantitative PCR was: 50℃ pre-denaturation for 2 min; 95℃ pre-denaturation for 2 min; 95℃ denaturation for 15 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles, and fluorescence signal collection at 72℃.

[0107] 3. The experimental results are shown in Table 11.

[0108] Table 11 Specificity Verification II Results

[0109] Serial number Group CT mean in conclusion 1 HAV Not detected Negative 2 HIV-1 Not detected Negative 3 HTLV Not detected Negative

[0110] The results in Table 10 show that the primers provided by the present invention have good detection specificity and will not identify other RNA viruses.

[0111] Comparative Example 1

[0112] A total of three pairs of primers were designed for HIV-2 viral sequences, including the first pair of primers used in Example 1 and the second and third pairs of primers. The specific sequences are shown in Table 12.

[0113] Table 12 Primer sequence information

[0114]

[0115]

[0116] The detection experimental steps and reaction conditions are as in Example 3 above. The working concentration of HIV-2 positive control is 1×10 6 IU / ml, the results are shown in Table 13 below.

[0117] Table 13 Comparative Example 1 Test Results

[0118] Group Ct mean The first pair of primers 23.85 The second pair of primers 28.40 The third pair of primers 31.69

[0119] As can be seen from Table 13, for the same concentration of HIV-2 positive control, the detection Ct value of the first pair of primers is much lower than that of the second pair of primers and the third pair of primers, indicating that the first pair of primers has a better performance in the detection sensitivity of HIV-2 virus.

[0120] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer for rapid detection of HIV-II virus based on fluorescent quantitative PCR, characterized in that: It includes a forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO:

2.

2. A kit for rapid detection of HIV-II virus based on fluorescent quantitative PCR, characterized in that: Comprising the primer according to claim 1.

3. The kit according to claim 2, characterized in that: Also included are qPCR assay master mix and positive controls.

4. The kit according to claim 2, characterized in that: The qPCR detection premix includes 2×TSINGKE Master qPCR Mix-SYBR (+UDG).

5. The kit according to claim 2, characterized in that: The positive control substance includes a recombinant plasmid containing a DNA fragment of HIV-II virus.

6. Use of the primers according to claim 1 in preparing a kit for detecting HIV-II virus infection.

7. Use of the primers according to claim 1 in preparing a kit for HIV-II virus contamination during stem cell isolation or culture.

8. A method for rapid detection of HIV-II virus contamination in stem cell isolation or culture based on fluorescent quantitative PCR, characterized in that: The following steps are involved: Extracting total RNA from the sample to be tested, and performing reverse transcription to obtain cDNA of the sample to be tested; Using the cDNA of the sample to be tested as a template, the primers described in claim 1 are used for qPCR amplification, and judging whether there is HIV-II virus contamination in the sample to be tested according to the Ct value of the test result: A positive result is when the CT value is ≤35.0 and the curve has an obvious exponential growth curve, indicating that the sample to be tested is contaminated with HIV-II virus; A test result of CT>40 or no Ct value and no obvious amplification curve is considered a negative result, indicating that the sample to be tested is not contaminated by HIV-II virus.

9. The method according to claim 8, characterized in that: The reaction system of the qPCR amplification is 10 μl, including the following components: 2×TSINGKE MasterqPCRMix-SYBR (+UDG) 5 μl, 10 μM upstream primer 0.5 μl, 10 μM downstream primer 0.5 μl and sample RNA 4 μl.

10. The method according to claim 8, characterized in that: The reaction procedure of the qPCR amplification was as follows: pre-denaturation at 50°C for 2 min; pre-denaturation at 95°C for 2 min; denaturation at 95°C for 15 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 40 cycles, and collection of fluorescence signals at 72°C.

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