CN110964842B - Sample processing reagent for detecting mycobacterium tuberculosis nucleic acid, kit and mycobacterium tuberculosis nucleic acid amplification method - Google Patents
Sample processing reagent for detecting mycobacterium tuberculosis nucleic acid, kit and mycobacterium tuberculosis nucleic acid amplification method Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
Abstract
The invention provides a sample processing reagent for detecting mycobacterium tuberculosis nucleic acid, a kit and a method for amplifying the mycobacterium tuberculosis nucleic acid, wherein the sample processing reagent comprises digestion solution, tris-HCl solution and lysate; the digestive juice is an aqueous solution containing the following components in concentration: dithiothreitol 13-14g/L; 100-105g/L of sodium chloride; 2-3g/L of potassium chloride; 14-15.5g/L of disodium hydrogen phosphate; 2-3g/L of monopotassium phosphate; the lysate is an aqueous solution containing the following components in concentration: naOH 0.08-0.15w/v%; SDS 0.02-0.03w/v%. The sample treatment reagent for detecting the mycobacterium tuberculosis nucleic acid can extract the mycobacterium tuberculosis DNA from the sputum sample more quickly and effectively, effectively remove the interference of human DNA, completely inactivate the mycobacterium tuberculosis in the treatment process, effectively reduce the risk of personnel infection, and is safer and more efficient. The kit for detecting the mycobacterium tuberculosis nucleic acid has good sensitivity, specificity and accuracy, and can realize rapid detection of the mycobacterium tuberculosis nucleic acid.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a sample processing reagent for detecting mycobacterium tuberculosis nucleic acid, a kit and a method for amplifying the mycobacterium tuberculosis nucleic acid.
Background
Tuberculosis (TB) is caused by Mycobacterium Tuberculosis (MTBC) and has been a global public health challenge for decades. It is estimated that 1040 ten thousand cases of tuberculosis are newly increased worldwide in 2016, and 170 ten thousand deaths are observed. Traditional tuberculosis diagnosis relies fundamentally on Acid Fast Bacilli (AFB) smear microscopy and selection of mycobacterial culture on solid media, but these methods have limitations. First, AFB smears have low sensitivity (30-40%) and their accuracy tends to be limited. Second, although culture-based methods have high accuracy and have long been considered as the gold standard for laboratory tuberculosis diagnosis, the long turn-around time of mycobacterial culture (3-8 weeks) often results in delayed diagnosis.
Commercially available real-time Polymerase Chain Reaction (PCR) detection methods, such as Abbott real-time MTB assay and GeneXpert MTB/RIF Ultra, can provide timely and accurate diagnosis of tuberculosis. However, these commercial tests are still too expensive to be routinely used, especially in most developing countries where the tuberculosis burden is high.
Disclosure of Invention
Based on this, the invention provides a sample processing reagent for detecting mycobacterium tuberculosis nucleic acid, a kit and a method for amplifying mycobacterium tuberculosis nucleic acid. The sample processing reagent for detecting the mycobacterium tuberculosis nucleic acid can extract the mycobacterium tuberculosis and DNA thereof from a sputum sample more quickly, effectively remove interference of human DNA, completely inactivate the mycobacterium tuberculosis, and is safer and more efficient.
The specific technical scheme is as follows:
a sample processing reagent for detecting a mycobacterium tuberculosis nucleic acid, the processing reagent comprising a digestion solution, a Tris-HCl solution and a lysate; the digestive juice is an aqueous solution containing the following components in concentration: dithiothreitol 13-14g/L; 100-105g/L of sodium chloride; 2-3g/L of potassium chloride; 14-15.5g/L of disodium hydrogen phosphate; 2-3g/L of monopotassium phosphate; the concentration of the Tris-HCl solution is 0.08-0.12mol/L; the lysate is an aqueous solution containing the following components in concentration: naOH 0.08-0.15w/v%; SDS 0.02-0.03w/v%.
In some of these embodiments, the digestive juice has a pH of 7.2 to 7.6.
In some of these embodiments, it is preferred that the digestive juice is an aqueous solution containing the following concentration components: dithiothreitol 13.33g/L; 104g/L of sodium chloride; potassium chloride 2.67g/L; 14.93g/L disodium hydrogen phosphate; 2.67g/L of monopotassium phosphate.
In some of these embodiments, the Tris-HCl solution has a concentration of 0.1.+ -. 0.01mol/L.
In some of these embodiments, it is preferred that the lysate is an aqueous solution containing the following concentration components: naOH 0.1w/v%; SDS0.025w/v%.
The invention also provides a sample processing method for detecting the mycobacterium tuberculosis nucleic acid.
The specific technical scheme is as follows:
a sample processing method for detecting a mycobacterium tuberculosis nucleic acid, comprising the steps of:
(1) Adding the digestive juice into a sputum sample, shaking and uniformly mixing, and then incubating for 10-15 minutes at 60-70 ℃;
(2) Centrifuging the mixed solution obtained in the step (1) at 13200 g+/-50 g for 8-15 minutes, and discarding the supernatant;
(3) Adding the Tris-HCl solution into the precipitate obtained in the step (2), shaking and uniformly mixing, centrifuging at 13200 g+/-50 g for 8-15 minutes, and discarding the supernatant;
(4) Adding the lysate to the precipitate obtained in the step (3), and then incubating for 45-60 minutes at 60-70 ℃;
(5) And (3) adding the Tris-HCl solution into the mixed solution obtained in the step (4), and uniformly mixing to obtain a DNA solution.
In some embodiments, the volume ratio of sputum sample, digestive juice, and lysate is 1: (0.1-0.3): (0.1-0.2); preferably, the volume ratio of the sputum sample, the digestive juice and the lysate is 1:0.1:0.1, the inventors found in the study that when the volume ratio of the sputum sample, the digestive juice and the lysate is 1:0.1: a better treatment effect can be obtained at 0.1.
In some of these embodiments, the incubation in step (1) is at 65 ℃ for 10 minutes.
In some of these embodiments, the centrifugation is performed at 13200g for 10 minutes in step (2); centrifugation is performed at 13200g for 10 minutes in step (3).
In some of these embodiments, the incubation in step (4) is at 65 ℃ for 45 minutes.
According to the sample processing method for detecting the mycobacterium tuberculosis nucleic acid, the viscous sputum is liquefied in a short time through the mild strong reducing agent digestive juice, so that bacteria and human cell fragments are separated, and human cells are not required to be completely dissolved, so that the sample processing method does not contain human DNA; further, after the sputum is liquefied and centrifuged, the supernatant is removed, tris-HCl solution with a pH value suitable for the physiological condition of the human body is added, and the residual human body cells and other impurities in the sample can be removed again; finally, adding the lysate, dissolving the cell wall of the bacteria to destroy and open the cell membrane, and releasing DNA.
The invention also provides a kit for detecting the mycobacterium tuberculosis nucleic acid.
The specific technical scheme is as follows:
a kit for detecting a mycobacterium tuberculosis nucleic acid, comprising the following components: the sample processing reagent for detecting a Mycobacterium tuberculosis nucleic acid; primer pair 1 for IS 6110; primer pair 2 for IS 6110; a probe primer directed against IS 6110; a quality control template; a probe primer for a quality control template;
the primer pair 1 for IS6110 comprises IS6110-FW1 with a sequence shown as SEQ ID NO.1 and IS6110-RV1 with a sequence shown as SEQ ID NO. 2; primer pair 2 for IS6110 comprises IS6110-FW2 with a sequence shown as SEQ ID NO.3 and IS6110-RV2 with a sequence shown as SEQ ID NO. 4; the sequence of the probe primer aiming at IS6110 IS shown as SEQ ID NO.5, the 5 'end of the probe primer IS modified with a fluorescent group, and the 3' end IS modified with a quenching group;
the quality control template contains a mouse RAB3A oncogene fragment and an IS6110 primer pair 2 binding region; the quality control template sequence is shown as SEQ ID NO. 6; the sequence of the probe primer aiming at the quality control template IS shown as SEQ ID NO.7, a fluorescent group IS modified at the 5 'end of the probe primer, a quenching group IS modified at the 3' end of the probe primer, and the fluorescent group and the quenching group are different from those of the probe primer aiming at IS 6110.
In some of these embodiments, the probe primer for IS6110 IS modified at the 5 'end with a fluorescent group FAM and at the 3' end with a quenching group BHQ1; the 5 'end of the probe primer aiming at the quality control template is modified with a fluorescent group LC610, and the 3' end is modified with a quenching group BBQ.
SEQ ID NO.1：5’-CCGGCCAGCACGCTAATTAACGGTTC-3’
SEQ ID NO.2：5’-TGTGGCCGGATCAGCGATCGTGGT-3’
SEQ ID NO.3：5'-CTGCACACAGCTGACCGA-3'
SEQ ID NO.4：5’-CGTTCGACGGTGCATCTG-3’
SEQ ID NO.5：5'-FAM-ATGGCGAACTCAAGGAGCACATCAGC-BHQ1-3'
SEQ ID NO.6：5’-GGTACCTCGCGAATGCATCTAGATGACCCAATTCGAGTCGATCTGCACACAGCTGACCGATCAAGGTACTGGGCCTGTGAAGTTTTCGAACTAGAAAGAGAAATGGGTGGAAGAGGCTAAGCCTGGCTTCCCGGAGCAGGCACAACATGCCTGTATGGGAAGGTTGGTATAGGCAGAGTCGTGACAGTGGTGCGTGATCGCCCCTTAGAGGAACTGTGGCTGTCACTGCAGGCTGAGCTCAGATGCACCGTCGAACGGCTGATGACCAAAATTGGGATCGGATCCCGGGCCCGTCGACTGCAGAGGCCTGCATGCAAGCTTGGCGTAATCATGGTCATAGCTGTTTC-3’
SEQ ID NO.7：5’-LC610-TATAGGCAGAGTCGTGACAGTGGTGC-BBQ-3’
In some of these embodiments, the kit for detecting a mycobacterium tuberculosis nucleic acid further comprises the following components: probe PCR Master Mix and DEPC water.
The invention also provides a nucleic acid amplification method of the mycobacterium tuberculosis, which is a single-step nested PCR amplification method, can detect two groups of primers with different annealing temperatures in a single tube at the same time, has good sensitivity, specificity and accuracy, and can realize rapid detection of the mycobacterium tuberculosis nucleic acid.
The specific technical scheme is as follows:
a method for amplifying a nucleic acid of mycobacterium tuberculosis, comprising the steps of:
(1) Collecting a sputum sample, and processing by using the sample processing method for detecting the mycobacterium tuberculosis nucleic acid to obtain a DNA solution;
(2) And (3) performing PCR amplification detection on the DNA solution obtained in the step (1) by using the kit for detecting the mycobacterium tuberculosis nucleic acid.
In some of these embodiments, the amplification system of the method for nucleic acid amplification of mycobacterium tuberculosis comprises the following components: IS6110-FW 1.8-1 mu M; IS6110-RV 1.8-1 mu M; IS6110-FW2 40-41 mu M; IS6110-RV2 40-41 mu M; 3.5-4 mu M of IS6110 probe primer; and 3.5-4 mu M of a quality control template probe primer.
In some of these embodiments, it is preferred that the amplification system of the method of detecting a mycobacterium tuberculosis nucleic acid comprises the following components: IS6110-FW 1.9 mu M; IS6110-RV 1.9 mu M; IS6110-FW 2.5 mu M; IS6110-RV 2.5 mu M; IS6110 probe primer 4 mu M; and 4 mu M of a quality control template probe primer.
In some of these embodiments, the amplification procedure of the method of detecting a mycobacterium tuberculosis nucleic acid is as follows: 95 ℃ for 2min;15 cycles: 95 ℃ for 5s and 72 ℃ for 30s;42 cycles: 95 ℃ for 5s and 6 ℃ for 30s, and collecting fluorescence at 6 ℃ for 30s; 30s at 40 ℃.
Compared with the prior art, the invention has the following beneficial effects:
1. the sample processing reagent for detecting the mycobacterium tuberculosis nucleic acid provided by the invention can extract the mycobacterium tuberculosis DNA from a sputum sample more quickly, and the extracted DNA has higher concentration and is more suitable for the detection of the next step. The sample treatment reagent is mild and good in effect, and can separate mycobacterium tuberculosis from human cell fragments in a short time without completely dissolving cells, so that the mycobacterium tuberculosis and human DNA can be distinguished more easily, and the interference of human DNA can be effectively removed. The sample treatment reagent can effectively reduce the denaturation of biomolecules, has a pH value similar to that of a human body, can more effectively dissolve proteins and remove the proteins, and ensures that the purity of the extracted DNA is higher. In addition, the sample treatment reagent completely inactivates mycobacterium tuberculosis in the treatment process, effectively reduces the risk of personnel infection, and is safer and more efficient.
2. The kit for detecting the mycobacterium tuberculosis nucleic acid provided by the invention can accurately detect the mycobacterium tuberculosis nucleic acid in the sample, and has the advantages of lower price and easy reagent acquisition. The amplification primers and probes provided in the kit are obtained by the inventor through a great deal of research and optimization, and can be used for simultaneously detecting the external primers with higher melting temperature and the internal primers with lower melting temperature in a single tube, so that the detection sensitivity and the detection specificity are improved. The amplification of the external primer and the internal primer is carried out in a single tube, and the PCR amplification product does not need to be manually operated, thereby reducing the possibility of cross contamination. The kit also contains a homologous internal quality control template, can more effectively monitor the condition of a PCR link sample, can be easily distinguished from a true negative result by PCR inhibition, has the advantages of high sensitivity, high specificity, high accuracy and the like, and can rapidly realize amplification detection of the mycobacterium tuberculosis nucleic acid.
Detailed Description
In order that the invention may be understood more fully, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended claims. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It should be understood that the experimental methods in the following examples, in which specific conditions are not noted, are generally performed under conventional conditions or under conditions suggested by the manufacturer. The various reagents commonly used in the examples are all commercially available products.
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. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Example 1A sample processing reagent for detecting Mycobacterium tuberculosis nucleic acid
The sample processing reagent for detecting the mycobacterium tuberculosis nucleic acid comprises the following components: digestion solution, tris-HCl solution and lysate.
The digestive juice is an aqueous solution containing the following components in concentration: dithiothreitol 13.33g/L; 104g/L of sodium chloride; potassium chloride 2.67g/L; 14.93g/L disodium hydrogen phosphate; 2.67g/L of monopotassium phosphate; the pH value of the digestion liquid is 7.4.
The concentration of the Tris-HCl solution is 0.1+/-0.01 mol/L, and the pH value is 7.4.
The lysate is an aqueous solution containing the following components in concentration: naOH 0.1w/v%; SDS0.025w/v%.
Example 2A sample processing method for detecting Mycobacterium tuberculosis nucleic acid
The sample processing method for detecting a mycobacterium tuberculosis nucleic acid of the present embodiment processes a sputum sample using the sample processing reagent of the embodiment 1, and includes the following steps:
(1) Adding 100 mu L of digestive juice into 1mL of sputum sample, vortex shaking and mixing uniformly, and then incubating for 10 minutes at 65 ℃;
(2) Centrifuging the mixed solution obtained in the step (1) at 13200g for 10 minutes, and discarding the supernatant;
(3) Adding 100 mu L of Tris-HCl solution into the precipitate obtained in the step (2), stirring and mixing uniformly by vortex, centrifuging at 13200g for 10 minutes, and discarding the supernatant;
(4) Adding 100 mu L of lysate to the precipitate obtained in the step (3), and then incubating at 65 ℃ for 45 minutes;
(5) And (3) adding 100 mu L of Tris-HCl solution into the mixed solution obtained in the step (4), and uniformly mixing to obtain a DNA solution, wherein the DNA solution can be used immediately or stored at the temperature of-20 ℃ for later use.
Example 3A kit for detecting Mycobacterium tuberculosis nucleic acid
The kit for detecting the mycobacterium tuberculosis nucleic acid comprises the following components: primer pair 1 for IS6110 for the sample processing reagent described in example 1; primer pair 2 for IS 6110; a probe primer directed against IS 6110; a quality control template; a probe primer for a quality control template; quantiNova Probe PCR Master Mix and DEPC water.
The primer pair 1 for IS6110 comprises IS6110-FW1 with a sequence shown as SEQ ID NO.1 and IS6110-RV1 with a sequence shown as SEQ ID NO. 2; primer pair 2 for IS6110 comprises IS6110-FW2 with a sequence shown as SEQ ID NO.3 and IS6110-RV2 with a sequence shown as SEQ ID NO. 4; the sequence of the probe primer aiming at IS6110 IS shown as SEQ ID NO.5, the 5 'end of the probe primer IS modified with a fluorescent group FAM, and the 3' end IS modified with a quenching group BHQ1;
the quality control template sequence is shown as SEQ ID NO. 6; the sequence of the probe primer aiming at the quality control template is shown as SEQ ID NO.7, the 5 'end of the probe primer aiming at the quality control template is modified with a fluorescent group LC610, and the 3' end is modified with a quenching group BBQ.
The quality control template (IC template) IS pUC57 plasmid containing mouse Rab3a oncogene and mycobacterium tuberculosis IS6110 sequence, and can be expressed in DH5 alpha colibacillus strain. The preparation method comprises the following steps: plasmid DNA was extracted by QIAprep Spin Miniprep kit (Qiagen Pte ltd.; catalog No. 27104); the plasmid DNA was PCR amplified to obtain a 346bp quality control template, which was then purified by a QIAquick PCR purification kit (Qiagen Pte ltd., catalog No. 28106); by dilution with EB buffer to 5X 10 -9 ng/. Mu.l to prepare working concentration of IC template. The method comprises the following steps:
A. extraction of plasmids
1. Frozen stock of DH 5. Alpha. IS6110 IC was subcultured with 12. Mu.l of 50mg/ml ampicillin in 6ml LB broth (DH 5. Alpha.) and incubated overnight at 37℃with shaking at 250 rpm.
2.6 ml of DH5α overnight broth was transferred to a 15ml tube and centrifuged at 3,000Xg for 5 minutes.
3. Bacteria were resuspended in 200. Mu.l of P1 buffer (RNase A added and stored at 4℃before use).
4. 200 μl of buffer P2 (SDS-lysis and NaOH denaturation) was added and gently inverted 6 times, followed by 280 μl of buffer N3 (neutralization) and immediately gently inverted 10 times.
5. Centrifuge at 13,000rpm for 10 minutes.
6. The QIAprep adsorption column was labeled and then the supernatant was added to the column.
7. Centrifuge at 13,000rpm for 1 minute (replace one collection tube, add the remaining supernatant and centrifuge again if necessary).
8. The collection tube was replaced and 650 μl buffer PE (ethanol added) was added.
9. Centrifuge at 13,000rpm for 1 minute.
10. The collection tube was replaced and centrifuged at 13,000rpm for 1 minute.
11. Discard the collection tube and replace the new 1.5ml Ep tube
12. 30ul of EB buffer was added to the adsorption column. Incubate for 1 min at room temperature and then centrifuge at 13,000rpm for 1 min.
13. Before use, please store at-20 ℃.
PCR amplified plasmid to IC template inventory
1. The PCR reaction mixtures were prepared as shown in table 1:
TABLE 1 PCR reaction mixtures
* Provided by HotStarTaq Plus DNA Polymerase units (Qiagen Pte Ltd., cat no: 203605)
a TBICplasmidF:5’-GGTACCTCGCGAATGCATCTAG-3’
b TBICplasmidR:5’-GAAACAGCTATGACCATGATTACGC-3’
2. Mu.l of the extracted plasmid DNA was added to the mixture.
3. PCR amplification was performed in a thermocycler under the conditions of table 2:
TABLE 2 PCR amplification conditions
4. Mu.l of 6 Xload dye was added to parafilm waxed paper.
5. The PCR products were pulsed. Mu.l of PCR product was transferred to a dye added dropwise to a 6X sample and mixed.
6. The mixture was transferred to wells of a 2% agarose gel.
7. Mu.l of a 100bp molecular size marker was added to the first well of the gel.
8. The gel was run at 120V for 38 minutes.
9. The gel was stained with ethidium bromide for 20 minutes with gentle shaking.
10. The gel was inspected under uv irradiation and the target band size of the IC template: 346bp.
C. Purifying IC template inventory and preparing working concentrations
1. To 1 volume (45. Mu.l) of the PCR product was added 5 volumes (225. Mu.l) of PB solution and mixed well.
2. The mixture was applied to a QIAquick adsorption column and centrifuged at 13,000rpm for 1 min.
3. The centrifuged liquid is discarded and the column is then placed in a new collection tube.
4. 750 μl of buffer PE was added to the column and centrifuged at 13,000rpm for 1 min.
5. The centrifuged liquid is discarded and the column is then placed in a new collection tube.
6. The column was again adsorbed at 13,000rpm for 1 minute. The collection tube is discarded. The column was placed in a clean 1.5ml Ep tube.
7. Mu.l of EB buffer was added to the center of the column.
8. The column was kept to stand for 1 minute. Centrifuge at 13000rpm for 1 minute.
9. The centrifuged liquid was collected as purified IC template stock and stored at-80℃for long term storage.
10. Spectrophotometry was performed using a Nanodrop instrument.
11. Dilution of IC templates with buffer EB to 5X 10 -7 ng/. Mu.l. Will be 5X 10 -7 ng/. Mu.l of IC template was stored in 20. Mu.l aliquots at-20 ℃. In each run, 5×10 should be prepared again -9 ng/. Mu.l working IC template.
The judgment criteria of the results of the above kit when used for detecting a Mycobacterium tuberculosis nucleic acid are shown in Table 3 below:
table 3 judgment Standard for results of kit
Note that: IS6110 positive: the sample has a ct value and an S-shaped curve; IS6110 negative: the samples have no ct values or no sigmoid curve. Positive quality control template: the sample has an S-shaped curve of ct value and IC; negative quality control template: sample IS6110 has no ct value or S-curve.
Example 4 a method for amplifying nucleic acid of mycobacterium tuberculosis the method for amplifying nucleic acid of mycobacterium tuberculosis of this example uses the kit of example 3 for amplification detection, comprising the steps of:
(1) Collecting a sputum sample, and processing by using the sample processing method for detecting the mycobacterium tuberculosis nucleic acid described in the embodiment 2 to obtain a DNA solution;
(2) Configuring a PCR amplification system and performing PCR amplification detection: a PCR amplification system was prepared as shown in Table 4, 5. Mu.L of the DNA solution obtained in step (1) was added to the system, and the mixture was mixed with shaking, followed by PCR amplification detection according to the amplification procedure shown in Table 5.
TABLE 4 PCR amplification System
TABLE 5 PCR amplification procedure
After the detection, the result was judged according to the judgment standard described in example 3.
Comparative example 1
The sample processing method for detecting the mycobacterium tuberculosis nucleic acid of the comparative example uses a conventional method to extract DNA of a sputum specimen, and comprises the following specific steps:
(1) The sputum is put into 4% NaOH with equal volume for liquefaction;
(2) Centrifuging the liquefied sample 13000 for 10min, discarding the supernatant, and adding 1mL of physiological saline;
(3) Adding a lysate into the bottom sediment, and carrying out metal bath for ten minutes at the temperature of 100 ℃;
(4) After the metal bath, the solution was centrifuged, and the supernatant was discarded, and DEPC water was added for dissolution to obtain a DNA solution.
Comparative example 2
The comparative example is a nucleic acid amplification method of mycobacterium tuberculosis, which is to use the GeneXpert MTB/RIF method for amplification detection, wherein a detection kit is MTB/RIF Assay purchased from Sairupi company, and the detection steps are carried out according to the specification, and specifically comprise the following steps:
(1) The sample is liquefied by adding twice as much sample processing reagent.
(2) Taking out 2mL of the liquefied sample, adding the liquefied sample into a kit, and loading the kit into a machine for detection.
DNA extraction was performed on 15 sputum samples using the sample treatment method for detecting Mycobacterium tuberculosis nucleic acid described in example 2 and comparative example 1, and concentration and purity of the extracted DNA solution were measured, and the results are shown in Table 6:
TABLE 6 determination of the concentration and purity of DNA solutions
| Sample number | Example 2 | Comparative example 1 |
| 1 | Concentration (ng/. Mu.l) | Concentration (ng/. Mu.l) |
| 2 | 3.2 | 2.8 |
| 3 | 6.4 | 5.4 |
| 4 | 13.2 | 10.1 |
| 5 | 2.5 | 2.1 |
| 6 | 6.8 | 5.9 |
| 7 | 28.5 | 25.3 |
| 8 | 32.1 | 26.8 |
| 9 | 16.2 | 14.2 |
| 10 | 15.2 | 13.2 |
| 11 | 14.2 | 10.2 |
| 12 | 13.5 | 10.1 |
| 13 | 3.5 | 2.8 |
| 14 | 4.6 | 3.3 |
| 15 | 5.4 | 4.1 |
As is clear from the results shown in Table 6, the sample processing method for detecting Mycobacterium tuberculosis nucleic acid (example 2) provided by the invention can successfully extract DNA in a sputum sample, and compared with the existing sample processing method for detecting Mycobacterium tuberculosis nucleic acid (comparative example 1), the sample processing method for detecting Mycobacterium tuberculosis nucleic acid has higher concentration of extracted nucleic acid and is more suitable for the detection of the next step.
39 sputum samples were tested using the method for detecting Mycobacterium tuberculosis nucleic acid described in example 4 and comparative example 2, and the results are shown in Table 7:
TABLE 7 Mycobacterium tuberculosis nucleic acid detection results
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As can be seen from the detection results in the above table, the detection effect of the nucleic acid amplification method (example 4) of Mycobacterium tuberculosis provided by the invention is equivalent to that of the conventional GeneXpert MTB/RIF method (comparative example 2), but the cost of the detection method provided by the invention is lower than that of the GeneXpert MTB/RIF method, the cost of the detection method of one sputum sample is about 35 yuan, and the cost of the detection of one sputum sample by the GeneXpert MTB/RIF method is about 600 yuan. Therefore, the method for detecting the mycobacterium tuberculosis nucleic acid is easier to popularize and use.
The technical features of the above embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
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Claims (7)
1. A sample processing method for detecting a nucleic acid of mycobacterium tuberculosis, comprising the steps of:
(1) Adding digestive juice into the sputum sample, shaking and uniformly mixing, and then incubating for 10-15 minutes at 60-70 ℃; the digestive juice is an aqueous solution containing the following components in concentration: dithiothreitol 13.33g/L; 104g/L of sodium chloride; potassium chloride 2.67g/L; 14.93g/L disodium hydrogen phosphate; 2.67g/L of monopotassium phosphate;
(2) Centrifuging the mixed solution obtained in the step (1) at 13200g +/-50 g for 8-15 minutes, and discarding the supernatant;
(3) Adding Tris-HCl solution into the precipitate obtained in the step (2), shaking and uniformly mixing, centrifuging at 13200g +/-50 g for 8-15 minutes, and discarding the supernatant; the concentration of the Tris-HCl solution is 0.08-0.12mol/L;
(4) Adding a lysate to the precipitate obtained in the step (3), and then incubating at 60-70 ℃ for 45-60 minutes; the lysate is an aqueous solution containing the following components in concentration: naOH 0.1w/v%; SDS0.025 w/v%;
(5) Adding Tris-HCl solution into the mixed solution obtained in the step (4), and uniformly mixing to obtain a DNA solution; the concentration of the Tris-HCl solution is 0.08-0.12mol/L.
2. The method for sample processing for detecting a Mycobacterium tuberculosis nucleic acid according to claim 1, wherein the concentration of Tris-HCl solution in the step (3) and/or the step (5) is 0.1mol/L.
3. The method for processing a sample for detecting a nucleic acid of mycobacterium tuberculosis according to claim 1, wherein the volume ratio of the sputum sample, the digestive juice and the lysate is 1: (0.1-0.3): (0.1-0.2).
4. The method for processing a sample for detecting a nucleic acid of Mycobacterium tuberculosis according to claim 3, wherein the volume ratio of the sputum sample, the digestive juice and the lysate is 1:0.1:0.1.
5. the method for processing a sample for detecting a nucleic acid of mycobacterium tuberculosis according to claim 1, wherein the incubation in step (1) is performed at 65 ℃ for 10 minutes.
6. The method for processing a sample for detecting a nucleic acid of mycobacterium tuberculosis according to claim 1, wherein the step (2) is performed for 10 minutes by centrifugation at 13200 and g; the solution was centrifuged at 13200 and g for 10 minutes in the step (3).
7. The method for processing a sample for detecting a nucleic acid of mycobacterium tuberculosis according to claim 1, wherein the incubation in step (4) is performed at 65 ℃ for 45 minutes.
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