CN117512212A - Multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA and detection method - Google Patents
Multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA and detection method Download PDFInfo
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Abstract
The invention discloses a multienzyme isothermal amplification kit for detecting HTLV-1 proviral DNA and a detection method. The multienzyme isothermal amplification kit comprises primer probe mixed solution, A buffer, freeze-dried enzyme powder, B buffer, lateral flow chromatography test strips, a positive control object and a negative control object. The primer probe mixed solution comprises a specific primer pair and a probe for detecting the HTLV-1 proviral DNA; the primer pair and the probe sequences are shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3. The invention combines the MIRA detection method with the gel Jin Celiu chromatography technology, can realize the visualization of the detection result by naked eye observation of the strip, can effectively detect the HTLV-1 proviral DNA, and can be used for the rapid in vitro diagnosis of HTLV-1 infection. The kit has the advantages of high sensitivity, good specificity, simple and portable operation and short time, and has high application value in basic medical institutions or blood centers (blood stations) with deficient laboratory resources.
Description
Technical Field
The invention belongs to the technical field of molecular detection, and particularly relates to a kit for detecting HTLV-1 proviral DNA, and particularly relates to a multienzyme isothermal amplification kit for detecting HTLV-1 proviral DNA and a detection method.
Background
Human T-cell leukemia virus (Human T-lymphotropic virus, HTLV) was the earliest one found in Human retroviruses, most commonly in HTLV-1 form. Humans often show asymptomatic infection after infection with HTLV-1, with latency periods up to 20 years, and later stages may cause adult T-cell leukemia, HTLV-related myelopathy/tropical spastic paraplegia, and various neurological diseases, with severe cases leading to death. HTLV is transmitted primarily by breast feeding, sexual contact and blood pathways.
At present, an enzyme-linked immunosorbent assay (ELISA) method is mainly adopted by domestic blood collection institutions to detect HTLV antibodies. However, antibody screening has some disadvantages, such as a longer detection window, and is prone to missed detection; ELISA methods are prone to more false positive results. The HTLV proviral DNA assay is a method for confirming a positive sample screened by an HTLV antibody, and has higher specificity than ELISA, so that it is necessary to develop a reagent for detecting HTLV proviral DNA.
The molecular diagnosis technology represented by real-time fluorescence quantitative PCR is considered as a powerful pathogen diagnosis tool due to high sensitivity and high specificity, but has strict requirements on the technical level of experimental instruments, environments and operators, is complex and time-consuming to operate, and is not suitable for popularization and application in basic medical institutions. The multi-enzyme isothermal rapid amplification (multienzyme isothermal rapid amplification, MIRA) technology is a novel nucleic acid rapid isothermal amplification technology. The technology uses 4 core proteins (recombinase, DNA helicase, single-stranded binding protein and DNA polymerase) to amplify target genes at a constant temperature of 25-42 ℃. The method can be completed within 5-20min, is simple and quick to operate, has low requirements on operators, does not need large-scale precise instruments and equipment, and can be carried out by only one water bath kettle or thermostat. Furthermore, the MIRA technique is combined with the gel Jin Celiu chromatography technique, and the detection result can be visualized by visually observing the strip. Compared with the PCR method, the MIRA technology has the advantages of high sensitivity, strong specificity, rapidness, portability, simple operation, visual result, suitability for on-site rapid detection and the like.
Disclosure of Invention
Aiming at the problems in the background technology, the invention provides a multienzyme isothermal amplification kit for detecting HTLV-1 proviral DNA and a detection method. The invention establishes a lateral flow chromatography test strip detection method for rapidly detecting HTLV-1 proviral DNA based on MIRA technology, greatly reduces requirements on environment, equipment and operators while ensuring high sensitivity and specificity, has simple and rapid operation, and has high application value in basic medical institutions or blood centers (blood stations) with deficient laboratory resources.
The technical scheme of the invention is as follows:
1. multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA
The multienzyme isothermal amplification kit comprises primer probe mixed solution, A buffer, freeze-dried enzyme powder, B buffer, lateral flow chromatography test strips, a positive control object and a negative control object.
The primer probe mixed solution comprises a specific primer pair and a probe for detecting HTLV-1 proviral DNA; the specific primer pair for detecting the HTLV-1 proviral DNA consists of an upstream primer and a downstream primer for detecting the HTLV-1 proviral DNA, wherein the sequence of the upstream primer for detecting the HTLV-1 proviral DNA is shown as SEQ ID NO.1, the sequence of the downstream primer is shown as SEQ ID NO.2, and the sequence of the probe is shown as SEQ ID NO. 3.
The 5' end of the primer shown in SEQ ID NO.2 is marked with a biotin group; the 5' end of the probe shown in SEQ ID NO.3 is marked with a FAM fluorescent group, a dSpacer (tetrahydrofuran, THF) is marked at a position 30nt from the 5' end, and the 3' end is marked with a modification group C3-spacer.
The positive control is a plasmid containing the HTLV-1 specific amplified gene fragment, wherein the nucleotide sequence of the HTLV-1 specific amplified fragment is shown as SEQ ID NO. 4.
The negative control is any one of DEPC (diethyl pyrocarbonate) treated water, deionized water and sterile water.
The freeze-dried enzyme powder comprises recombinase, DNA helicase, single-chain binding protein and DNA polymerase.
The buffer A comprises constant-temperature amplification buffer solution and dNTP; the B buffer comprises a magnesium acetate solution.
2. MIRA-based HTLV-1 proviral DNA detection method
The HTLV-1 proviral DNA detection method adopts the multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA, and comprises the following steps:
1) Extracting genomic DNA of a blood sample to be detected by using a genomic DNA extraction kit to obtain a sample genomic DNA extract;
2) Preparing MIRA reaction liquid by utilizing primer probe mixed liquid, A buffer, freeze-dried enzyme powder and B buffer in a multienzyme isothermal amplification kit, respectively placing a sample genome DNA extract, a positive control object and a negative control object into corresponding MIRA reaction liquid to obtain sample detection liquid, positive detection liquid and negative detection liquid, and then placing the three detection liquids into a water bath kettle or a thermostat for reaction;
3) And detecting the reacted sample detection liquid, positive detection liquid and negative detection liquid by adopting a lateral flow chromatography test strip, and obtaining the color development results of the three types of lateral flow chromatography test strips after visually observing the color development conditions of a quality control line and a detection line of the lateral flow chromatography test strip, thereby obtaining the detection result of the HTLV-1 proviral DNA.
In the step 2), when the sample detection liquid, the positive detection liquid and the negative detection liquid are subjected to MIRA reaction in a water bath kettle or a thermostat, the reaction temperature is 37-38 ℃ and the reaction time is 15-20min.
In the step 2), at least one sample genomic DNA extract is used, and the positive control and the negative control are used in one sample.
In the step 3), when the quality control line in the color development result corresponding to the negative detection liquid is blue, the detection line is colorless, and the quality control line in the color development result corresponding to the positive detection liquid is blue, the detection line is red, the experiment is effective; otherwise, the experiment is regarded as invalid, and the experiment needs to be repeated; under the experimental effect, in the color development results corresponding to the sample genome DNA extract, when the quality control line is blue and the detection line is red, the sample genome DNA extract is judged to be positive; when the quality control line is blue and the detection line is colorless, the detection line is judged to be negative; and when the quality control line and the detection line are colorless, judging that the test is invalid, and repeating the test.
The method specifically comprises the following steps:
(1) The kit does not contain a genome DNA extraction reagent, and a blood sample can use a commercially available genome DNA extraction kit, such as a blood genome DNA extraction kit (Tiangen Biochemical technology Co., beijing, china), and the specific extraction steps refer to the instruction of the extraction kit to obtain n-3 sample genome DNA extracts.
(2) The total volume of each reaction was 50. Mu.l, and 50. Mu.l. Times.n MIRA reaction solution was prepared for the number of reaction samples n (number of reaction samples=number of samples to be examined+number of negative controls 1+number of positive controls 1+1): specifically, A buffer n×29.4. Mu.L, primer probe mixture n×4.6. Mu.L, sterile ddH were taken 2 O n ×8.5 μl was mixed in a centrifuge tube, centrifuged at low speed for several seconds, and dispensed into lyophilized enzyme powder reaction tubes at 42.5 μl/tube.
(3) Taking n-3 parts of sample genome DNA extract, 1 part of negative control substance and 1 part of positive control substance, respectively adding 5 mu L of each of the sample genome DNA extract, 1 part of negative control substance and 1 part of positive control substance into corresponding freeze-dried enzyme powder reaction tubes, shaking and mixing uniformly, centrifuging at a low speed for a plurality of seconds, then adding 2.5 mu L of B buffer into each reaction tube, fully mixing the mixture after upside down, immediately placing the mixture into a water bath pot or a thermostat, and incubating at 37-38 ℃ for 15-20min.
(4) After the reaction is finished, diluting the sample detection liquid, the positive detection liquid and the negative detection liquid by 10-20 times, inserting the sample end of the lateral flow chromatography test strip into a centrifuge tube, balancing at room temperature, and observing a quality control line and a detection line within 10min to obtain a corresponding color development result.
(5) When the quality control line in the color development result corresponding to the negative detection liquid is blue, the detection line is colorless, and the quality control line in the color development result corresponding to the positive detection liquid is blue, the detection line is red, the experiment is effective; otherwise, the experiment is regarded as invalid, and the experiment needs to be repeated; under the experimental effect, in the color development results corresponding to the sample genome DNA extract, when the quality control line is blue and the detection line is red, the sample genome DNA extract is judged to be positive; when the quality control line is blue and the detection line is colorless, the detection line is judged to be negative; and when the quality control line and the detection line are colorless, judging that the test is invalid, and repeating the test.
The invention is particularly suitable for on-site rapid screening of pathogenic microorganisms, can provide diagnosis basis at the first time and improves detection efficiency. The multi-enzyme isothermal amplification kit and the detection method for detecting the HTLV-1 proviral DNA can realize rapid and accurate identification of the HTLV-1 in blood, improve the efficiency of detecting the HTLV-1, are simple, rapid and portable, solve the problems of complicated operation flow, dependence on large-scale precise equipment (such as a PCR instrument), low detection efficiency, time consumption and the like in the prior art, are particularly suitable for basic level and field detection, and have wide application prospects.
The invention has the beneficial effects that:
(1) Detection efficiency can be improved: the whole MIRA test process only needs 15-20min, which is far lower than the qPCR test time of 60min, greatly shortens the amplification time and improves the detection efficiency.
(2) The reaction temperature can be reduced: MIRA only needs to be constant at 37-38 ℃ to complete the test, and the temperature is far lower than 60-95 ℃ of qPCR.
(3) The operation is simple and the visualization is carried out: the requirements on operators are low, large-scale precise instruments and equipment are not needed, and the experiment can be carried out by one water bath kettle or thermostat, so that the method is suitable for rapidly screening pathogenic microorganisms on site; the amplification products were detected using lateral flow chromatography test strips, and the results were visualized.
(4) High sensitivity and high specificity: by optimizing the reaction time and temperature, the sensitivity and specificity of the detection method are improved.
Drawings
FIG. 1 is a graph showing the results of the detection of HTLV-1 optimal reaction temperature in accordance with the present invention;
FIG. 2 is a graph showing the results of the detection of HTLV-1 in the present invention;
FIG. 3 is a graph showing the sensitivity results of the kit of the invention for detecting HTLV-1 standard;
FIG. 4 is a graph showing the specificity results of the kit of the present invention, 1: HTLV-1;2: HBV;3: HCV;4: HEV;5: CMV;6: HSV;7: b19;8: an EBV;9: a negative control; 10: positive control.
Detailed Description
Embodiments of the present invention will be described in detail with reference to examples.
Example 1
Preparation of multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA
1. Design synthesis of primer probes
Downloading the gene sequence of HTLV-1 through NCBI gene library, performing multi-sequence comparison by DNAMAN software after downloading, screening a gene conservation region, designing a primer probe by using primer premier 5.0 according to MIRA primer probe design principle, inputting the designed primer probe sequence to NCBI BLAST for specific comparison, screening a primer probe capable of amplifying HTLV-1 and not carrying out specific amplification with other pathogens, detecting an upstream primer sequence of HTLV-1 proviral DNA as shown in SEQ ID NO.1, and detecting a downstream primer sequence as shown in SEQ ID NO.2, wherein the probe sequence is shown in SEQ ID NO. 3.
Wherein, the 5' end of the primer shown in SEQ ID NO.2 is marked with a biotin group; the 5' end of the probe shown in SEQ ID NO.3 is marked with a FAM fluorescent group, a dSpacer (tetrahydrofuran, THF) is marked at a position 30nt from the 5' end, and the 3' end is marked with a modification group C3-spacer.
The primers and probes were synthesized by the company Shanghai, inc.
2. Positive plasmid preparation
The HTLV-1 specific amplified gene fragment was connected to a plasmid vector pUC57 (division of biological engineering (Shanghai)) by molecular cloning, and after transformation, cultivation and identification, plasmid DNA was extracted, and the concentration of the plasmid DNA was determined by using a Nanodrop2000 ultramicro spectrophotometer, according to copies/. Mu.l= (6.02X10) 23 )×(ng/μL×10 -9 ) Calculation of/(DNAlength.times.660), determination of the copy number of plasmid DNA, and dilution to 10 7 copies/μl。
The nucleotide sequence of the HTLV-1 specific amplified fragment is shown as SEQ ID NO. 4.
Establishment of MIRA reaction System and determination of reaction temperature and reaction time
The MIRA reaction was performed with a DNA isothermal rapid amplification kit (colloidal gold type) (Fingered Anpu future Biotechnology Co., ltd.) comprising a reaction tube containing lyophilized enzyme powder, A buffer and B buffer.
The MIRA system is as follows: the total reaction volume was 50. Mu.l, wherein A buffer 29.4. Mu.l, primer probe mix 4.6. Mu.l, extracted genomic DNA template, negative control, positive control each 5. Mu.l, sterile ddH 2 O8.5μl，B Buffer 2.5μL。
To screen out the optimal reaction conditions, the reaction conditions were adjusted to 10 3 -10 1 The copies/. Mu.l plasmid was used as template and sterile ddH was used 2 O was used as a negative control and the MIRA system was evaluated at different temperatures (35, 37 and 39 ℃) and reaction times (10, 15, 20, 25 and 30 min). As a result, as shown in FIG. 1, the concentration was 10 at 35 ℃ 3 -10 1 Only the quality control lines appear for the plasmids of copies/. Mu.l and for the negative control; at 37 deg.C, the concentration is 10 3 The plasmids of the copies/μl are used as test strips of the amplification template, and the quality control line and the detection line are both developed; at 39 ℃ under 10 DEG C 3 -10 1 The copies/. Mu.l plasmid and negative control were presented simultaneously as a control line and a detection line, so 37℃was chosen as the temperature for the subsequent experiments. As shown in FIG. 2, the detection line for MIRA amplification for 15min showed significant color development, and the detection line for negative control was developed for 25min and above, so that 15min was selected as the optimal amplification time.
Optimized optimal reaction conditions: incubate at 37℃for 15min. After completion of the reaction, 8. Mu.L of the mixture was added to the reaction mixture to give a solution containing 152. Mu.L of sterile ddH 2 And (3) after uniformly mixing in the centrifuge tube of O, inserting the sample end of the lateral flow chromatography test strip into the centrifuge tube for balancing at room temperature, and observing the interpretation results of the quality control line and the detection line within 5min.
Test one: sensitivity evaluation of the kit of the invention
The sensitivity of the multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA described in example one was evaluated by the following specific method: concentration is set to 10 7 Sterile ddH for copies/. Mu.l of HTLV-1 positive plasmid 2 O was diluted 10 times to give 10 7 -10 1 The 7 dilutions of positive plasmid solution were amplified according to the isothermal amplification assay method optimized in example one using each of the 7 dilutions of positive plasmid solution as template, followed by detection of the amplified product using lateral flow chromatography test strips.
The results are shown in FIG. 3, 10 7 -10 3 Amplification products with copies/. Mu.l plasmid as template simultaneously appear as quality control line and detection line at a concentration of 10 2 -10 1 Only the quality control line appears in the copies/. Mu.l plasmid and the negative control, so the sensitivity of the kit for detecting HTLV-1 proviral DNA is 10 3 copies/μl。
And (2) testing II: specificity evaluation of the kit of the invention
The specificity of the kit for detecting HTLV-1 proviral DNA in the first embodiment was evaluated by the following specific method: the isothermal amplification test method optimized according to example one was used to test HTLV-1 positive samples and other viruses such as Hepatitis B Virus (HBV), hepatitis C Virus (HCV), hepatitis E Virus (HEV), human Cytomegalovirus (CMV), herpes Simplex Virus (HSV), human parvovirus (B19), epstein-Barr virus (EBV) positive samples, and negative and positive controls were set.
As shown in FIG. 4, only the amplification products of the HTLV-1 positive sample and the positive control show the quality control line and the detection line simultaneously, and the other 7 viruses and the negative control show the quality control line only, so that the kit has good specificity, can detect the HTLV-1 specifically, and has no cross reaction with other pathogens.
And (3) test III: detection of clinical samples by the kit of the invention
500 clinical blood samples were collected, genomic DNA of the samples was extracted using a commercially available genomic DNA extraction kit, and then genomic DNA extracted from 500 clinical samples was detected using the isothermal amplification kit according to the isothermal amplification detection method described in example one, with the following results: the clinical blood sample is 500 times, 1 part of HTLV-1 positive sample is screened out, and the sample accords with the fluorescent quantitative PCR detection result, so that the accuracy of the kit is verified.
The above embodiments are merely illustrative of the principles and applications of the present invention, and the scope of the invention is not limited to the above embodiments.
The gene sequence involved in the invention is as follows:
SEQ ID No.1；
name: upstream primer
The source is as follows: synthetic construct
Type (2): other DNA
5’-AGACCCCGGACTCCGGCCCCAAAACCTG-3’
SEQ ID No.2；
Name: downstream primer
The source is as follows: synthetic construct
Type (2): other DNA
5’-Biotin-GGCGTGCCATCGGTAAATGTCCAAATAA-3’
SEQ ID No.3；
Name: probe with a probe tip
The source is as follows: synthetic construct
Type (2): other DNA
5’-FAM-GCCAGCTCGGGGCCTTCCTCACCAATGTTC/THF/
CTACAAGCGAATAGA-3’C3spacer
SEQ ID No.4；
Name: HTLV-1 specific amplified fragment
The source is as follows: synthetic construct
Type (2): other DNA
AGACCCCGGACTCCGGCCCCAAAACCTGTACACCCTCTGGGGAGGCTCCGTTGTCTGCATGTACCTCTACCAGCTTTCCCCCCCCATCACCTGGCCCCTCCTGCCCCACGTGATTTTTTGCCACCCCGGCCAGCTCGGGGCCTTCCTCACCAATGTTCCCTACAAGCGAATAGAAGAACTCCTCTATAAAATTTCCCTCACCACAGGGGCCCTAATAATTCTACCCGAAGACTGTTTGCCCACCACCCTTTTCCAGCCTGCTAGGGCACCCGTCACGCTAACAGCCTGGCAAAACGGCCTCCTTCCGTTCCACTCAACCCTCACCACTCCAGGCCTTATTTGGACATTTACCGATGGCACGCC
<110> Zhejiang university medical college affiliated first hospital
<120> Multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA and detection method
<160>4
<170>SIPOSequenceListing 1.0
<210>1
<211>28
<212>DNA
<213> Artificial sequence (Artificial Sequence)
<400>1
agaccccgga ctccggcccc aaaacctg 28
<210>2
<211>28
<212>DNA
<213> Artificial sequence (Artificial Sequence)
<400>2
ggcgtgccat cggtaaatgt ccaaataa 28
<210>3
<211>45
<212>DNA
<213> Artificial sequence (Artificial Sequence)
<400>3
gccagctcgg ggccttcctc accaatgttc ctacaagcga ataga 45
<210>4
<211>363
<212>DNA
<213> Artificial sequence (Artificial Sequence)
<400>4
Claims (10)
1. The multi-enzyme isothermal amplification kit for detecting the HTLV-1 proviral DNA is characterized by comprising a primer probe mixed solution, abuffer, freeze-dried enzyme powder, B buffer, a lateral flow chromatography test strip, a positive control object and a negative control object.
2. The multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA according to claim 1, wherein the primer probe mixed solution comprises a specific primer pair and a probe for detecting HTLV-1 proviral DNA; the specific primer pair for detecting the HTLV-1 proviral DNA consists of an upstream primer and a downstream primer for detecting the HTLV-1 proviral DNA, wherein the sequence of the upstream primer for detecting the HTLV-1 proviral DNA is shown as SEQ ID NO.1, the sequence of the downstream primer is shown as SEQ ID NO.2, and the sequence of the probe is shown as SEQ ID NO. 3.
3. The kit for detecting the multiplex isothermal amplification of HTLV-1 proviral DNA according to claim 1, wherein the positive control is a plasmid containing an HTLV-1 specific amplified gene fragment, and wherein the nucleotide sequence of the HTLV-1 specific amplified fragment is shown as SEQ ID NO. 4.
4. The kit for the detection of HTLV-1 proviral DNA of claim 1, wherein the negative control is any of DEPC treated water, deionized water and sterile water.
5. The kit for the multiplex isothermal amplification for detecting HTLV-1 proviral DNA according to claim 1, wherein the lyophilized enzyme powder comprises a recombinase, a DNA helicase, a single stranded binding protein and a DNA polymerase.
6. The multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA according to claim 1, wherein the A buffer comprises isothermal amplification buffer and dNTPs; the B buffer comprises a magnesium acetate solution.
7. A method for detecting HTLV-1 proviral DNA based on MIRA, characterized in that the method for detecting HTLV-1 proviral DNA uses a multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA according to any one of claims 1-6, the method comprising the steps of:
1) Extracting genomic DNA of a blood sample to be detected by using a genomic DNA extraction kit to obtain a sample genomic DNA extract;
2) Preparing MIRA reaction liquid by utilizing primer probe mixed liquid, A buffer, freeze-dried enzyme powder and Bbuffer in a multienzyme isothermal amplification kit, respectively placing a sample genome DNA extract, a positive control object and a negative control object into corresponding MIRA reaction liquid to obtain sample detection liquid, positive detection liquid and negative detection liquid, and then placing the three detection liquids into a water bath kettle or a thermostat for reaction;
3) And (3) detecting the reacted sample detection liquid, positive detection liquid and negative detection liquid by adopting a lateral flow chromatography test strip to obtain the color development results of the three types of lateral flow chromatography test strips, thereby obtaining the HTLV-1 proviral DNA detection result.
8. The method for detecting HTLV-1 proviral DNA based on MIRA according to claim 7, wherein in 2), the reaction temperature is 37-38 ℃ and the reaction time is 15-20min when the sample detection solution, the positive detection solution and the negative detection solution are subjected to MIRA reaction in a water bath or a thermostat.
9. The method of claim 7, wherein in 2) at least one sample genomic DNA extract is used, and the positive control and the negative control are used.
10. The method for detecting HTLV-1 proviral DNA based on MIRA according to claim 7, wherein in the 3) the experiment is effective when the quality control line in the color development result corresponding to the negative detection liquid is blue, the detection line is colorless, and the quality control line in the color development result corresponding to the positive detection liquid is blue, the detection line is red; otherwise, the experiment is regarded as invalid, and the experiment needs to be repeated; under the experimental effect, in the color development results corresponding to the sample genome DNA extract, when the quality control line is blue and the detection line is red, the sample genome DNA extract is judged to be positive; when the quality control line is blue and the detection line is colorless, the detection line is judged to be negative; and when the quality control line and the detection line are colorless, judging that the test is invalid, and repeating the test.
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| CN202311479099.3A CN117512212A (en) | 2023-11-08 | 2023-11-08 | Multi-enzyme isothermal amplification kit for detecting HTLV-1 proviral DNA and detection method |
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