PCR Detection Composition for Mycobacterium tuberculosis and Its Tongue Swab Detection Kit

Abstract

The present invention relates to a PCR detection composition for Mycobacterium tuberculosis and a tongue swab detection kit thereof. The PCR detection composition for Mycobacterium tuberculosis includes a primer set for detecting the IS6110 sequence, which is shown by the nucleotide sequences SEQ ID NO: 1 to 3, a primer set for detecting the IS1081 sequence, which is shown by the nucleotide sequences SEQ ID NO: 4 to 5, and an internal standard primer set for tongue swab detection, which is shown by the nucleotide sequences SEQ ID NO: 7 to 9. It has the advantages of high sensitivity and good specificity, and can solve the problems such as insufficient detection sensitivity of tongue swabs with low bacterial load and poor specificity caused by various interfering substances and other microorganisms existing in tongue swabs. By using the tongue swab PCR detection reagent and detection kit of the present invention, rapid detection of Mycobacterium tuberculosis in tongue swab samples can be achieved, and the lowest detection limit can reach 10 CFU / mL.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to a PCR detection composition for Mycobacterium tuberculosis and a tongue swab detection kit thereof. Background Art

[0002] Tuberculosis is a systemic chronic infectious disease caused by Mycobacterium Tuberculosis Complex (MTC) transmitted through the respiratory tract, and is widely prevalent worldwide. Tuberculosis can affect multiple organs, and pulmonary tuberculosis is its main form. Therefore, sputum specimens are the primary detection type for pulmonary tuberculosis patients. However, infectious aerosols are generated during the collection of sputum specimens, which pose potential biosafety hazards to the environment and laboratory personnel. At the same time, it may be difficult to obtain sputum from young children, the elderly, patients without productive cough, and HIV-infected individuals, which increases the difficulty of diagnosing pulmonary tuberculosis in such populations. As a specimen with simple, rapid collection and relatively low biosafety risk, tongue swabs can be an ideal alternative to sputum.

[0003] Tongue swab collection is simple, rapid, and non-invasive, and can collect cell samples containing oral microorganisms. Research has shown that Mycobacterium tuberculosis DNA can be detected at a high frequency in the oral mucosa of adult patients with active pulmonary tuberculosis, providing the possibility for diagnosing pulmonary tuberculosis using molecular biology methods. The collection process of tongue swabs is relatively safe, with a low risk of generating aerosols, and is also suitable for patients without sputum, and its applicability is superior to sputum specimens to a certain extent.

[0004] Tongue swabs can not only provide a more convenient and reliable method for diagnosing pulmonary tuberculosis for patients without sputum, but also can be used for screening a large population. Most transmissions of pulmonary tuberculosis occur from the initial stage of the disease to the treatment period. Actively conducting active screening of high-risk populations can detect and treat pulmonary tuberculosis earlier, achieve early detection of pulmonary tuberculosis patients, effectively block its transmission in the community, and provide important technical support for the realization of the global goal of ending the tuberculosis epidemic by 2035.

[0005] Molecular diagnostic technology targets genes for detection. The qPCR technology is a relatively mature one in molecular diagnostic technologies. Based on two primers, a TaqMan probe is added. After binding to specific sites, the cleavage activity of Taq enzyme is used to release fluorescent groups. The machine collects fluorescence signals in real time and presents image results. The detection time is usually within 2 hours, and it has the characteristics of rapid detection, high sensitivity, and strong specificity.

[0006] At present, there are the following difficulties in the qPCR detection of tuberculosis by tongue swabs. First, the mycobacterium tuberculosis load on the tongue is relatively low, and both the sampling site and method may lead to insufficient detection sensitivity of tongue swabs. Second, mycobacterium tuberculosis has a complex cell wall structure, resulting in difficult cell wall breaking and nucleic acid release, thus affecting the detection effect of tongue swabs. Finally, there are many interfering substances and other microorganisms on the tongue surface, which requires the detection technology to have good specificity to ensure the accuracy of the detection results. Summary of the Invention

[0007] The object of the present invention is to provide a PCR detection composition for mycobacterium tuberculosis, which has the advantages of high sensitivity and good specificity, and can solve the problems of insufficient detection sensitivity of tongue swabs with low bacterial load and poor specificity caused by various interfering substances and other microorganisms in tongue swabs.

[0008] The present invention also provides a tongue swab PCR detection reagent, which uses tongue swab samples for detection and has the advantage of being able to quickly lyse mycobacterium tuberculosis in tongue swabs to release the nucleic acid template of mycobacterium tuberculosis, thereby shortening the sample detection time.

[0009] The technical solution adopted by the present invention to solve its technical problems is as follows:

[0010] A PCR detection composition for mycobacterium tuberculosis, the PCR detection composition includes the following primers and probes:

[0011] Primer set for detecting IS6110 sequence:

[0012] IS6110-F5, having the nucleotide sequence shown in SEQ ID NO:1,

[0013] IS6110-R5, having the nucleotide sequence shown in SEQ ID NO:2,

[0014] IS6110-P5, having the nucleotide sequence shown in SEQ ID NO:3,

[0015] Primer set for detecting IS1081 sequence:

[0016] IS1081-F7, having the nucleotide sequence shown in SEQ ID NO:4,

[0017] IS1081-R7, having the nucleotide sequence shown in SEQ ID NO:5,

[0018] IS1081-P7, having the nucleotide sequence shown in SEQ ID NO:6,

[0019] Internal standard primer set for tongue swab detection:

[0020] Actin-F3, having the nucleotide sequence shown in SEQ ID NO:7,

[0021] Actin-R3, having the nucleotide sequence shown in SEQ ID NO:8,

[0022] Actin-P3, having the nucleotide sequence shown in SEQ ID NO:9,

[0023] Among them, the specific probe has a 5'-end fluorescent group modification and / or a 3'-end quenching group modification.

[0024] Preferably, the fluorescent group is selected from any one of FAM, VIC, ROX, CY5; the quenching group is selected from BHQ1 or BHQ2.

[0025] Preferably, the probe IS6110-P5 and IS1081-P7 are labeled with FAM, and the probe Actin-P3 is labeled with CY5.

[0026] A tongue swab PCR detection reagent based on the PCR detection composition of Mycobacterium tuberculosis described in the present invention, the tongue swab PCR detection reagent includes a tongue swab nucleic acid rapid extraction reagent and a Mycobacterium tuberculosis detection reagent, wherein the tongue swab nucleic acid rapid extraction reagent includes: Triton X-100, Tris-HCl, EDTA, Chelex-100, digitonin and dodecyldimethylbetaine; the Mycobacterium tuberculosis detection reagent includes: the PCR detection composition of Mycobacterium tuberculosis described in the present invention, PCR buffer, dNTPs, hot start enzyme, UNG enzyme, reverse transcriptase, graphene oxide and betaine.

[0027] Preferably, the tongue swab nucleic acid rapid extraction reagent includes the following components at a final concentration: Triton X-100 1 wt%, Tris-HCl 10 mM, EDTA 1 mM, Chelex-100 0.5-2 wt%, digitonin 1-3 wt% and dodecyldimethylbetaine 1-3 wt%, and the pH is 7.0-8.0.

[0028] More preferably, the tongue swab nucleic acid rapid extraction reagent includes Chelex-100 1 wt%, digitonin 1.5 wt% and dodecyldimethylbetaine 2 wt% at a final concentration.

[0029] Preferably, the rapid nucleic acid extraction reagent for tongue swabs comprises the following components at final concentrations: Triton X-100 1 wt%, Tris-HCl 10 mM, EDTA 1 mM, Chelex-100 1 wt%, digitonin 1.5 wt%, and dodecyldimethylbetaine 2 wt%, with a pH of 7.0 - 8.0.

[0030] Preferably, the Mycobacterium tuberculosis detection reagent comprises the following components at final concentrations: the PCR detection composition for Mycobacterium tuberculosis of the present invention, 1xPCR buffer, Taq enzyme 6 U, MgCl2 3 mM, graphene oxide 1 - 2 ng / mL, and betaine 0.5 - 1 M.

[0031] Preferably, the detection method of this reagent comprises the following steps: Mix the rapid nucleic acid extraction reagent for tongue swabs with a tongue swab sample, heat it in a water bath at 95°C for 10 minutes, centrifuge at 12,000 r / min for 3 minutes, and take the supernatant to complete the rapid nucleic acid extraction. Using this gene detection method, the detection of Mycobacterium tuberculosis nucleic acid can be completed within 1 hour, greatly improving the detection efficiency.

[0032] Preferably, the sampling site of the selected tongue swab is the root of the tongue, and the sampling time each time is not less than 15 seconds.

[0033] A tongue swab detection kit, which contains the tongue swab PCR detection reagent of the present invention.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] 1. The PCR detection composition for Mycobacterium tuberculosis provided by the present invention is designed based on the specific target IS6110 sequence and IS1081 sequence of Mycobacterium tuberculosis. The dual-target detection can effectively reduce the probability of sample missed detection and misdetection, improve the accuracy of the kit detection. At the same time, using the Actin target as an internal standard can effectively monitor the nucleic acid release of the sample and the detection effect of the system, avoiding detection errors caused by non-standard tongue swab sampling.

[0036] 2. In view of the difficulty in breaking the cell wall of Mycobacterium tuberculosis, digitonin and dodecyldimethylbetaine are added to the rapid nucleic acid extraction reagent for tongue swabs. Experiments have proved that digitonin can act on the lipids on the cell membrane of Mycobacterium tuberculosis, causing the cell membrane to perforate and dissolve. Dodecyldimethylbetaine is a non-ionic surfactant that can effectively dissolve the lipids and proteins on the cell membrane, destroy the cell membrane, and then depolymerize the nucleoproteins in the cells. The present invention proves that the mixed use of the two at appropriate concentrations is more conducive to the release of nucleic acids from Mycobacterium tuberculosis. At the same time, the high-efficiency chelating effect of Chelex-100 inactivates nucleases, thereby protecting DNA molecules from degradation. By heating and centrifugation, denatured proteins and Chelex-100 are removed. The whole process has fewer steps, reduces the chance of contamination, and reduces the loss of nucleic acids. At the same time, adding appropriate concentrations of graphene oxide and betaine to the Mycobacterium tuberculosis detection reagent effectively improves the detection limit of Mycobacterium tuberculosis.

[0037] 3. Using the tongue swab PCR detection reagent and detection kit of the present invention, the rapid detection of Mycobacterium tuberculosis in tongue swab samples can be realized. After testing with clinical samples, it is found that the lowest detection limit can reach 10 CFU / mL, and there is no cross-reaction with various strains with high homology or similar clinical symptoms. The detection of nucleic acids of Mycobacterium tuberculosis can be completed within 1 hour. The method of the present invention greatly improves the detection efficiency of tongue swabs and helps to achieve the early detection of pulmonary tuberculosis patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Amplification curves of primers and probes in Example 1 and Comparative Examples 1 and 2;

[0039] Figure 2 Amplification curve of specific reaction in Example 2;

[0040] Figure 3 Amplification curve of nucleic acid extraction using Experimental Group 5 in Example 4;

[0041] Figure 4 Amplification curve of nucleic acid extraction using the column extraction method in Comparative Group 7 in Example 4;

[0042] Figure 5 Amplification curve of nucleic acid extraction using the magnetic bead method in Comparative Group 8 in Example 4;

[0043] Figure 6 Schematic diagram of different sampling sites in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0044] The technical solution of the present invention will be further specifically described below through specific embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any formal modification and / or change made to the present invention will fall within the protection scope of the present invention.

[0045] In the present invention, unless otherwise specified, all parts and percentages are in weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The methods in the following embodiments are all conventional methods in the art unless otherwise specified.

[0046] The reagents used in the following embodiments can be purchased from conventional biochemical reagent stores unless otherwise specified.

[0047] Among them, the standard strains were purchased from Beina Biotechnology.

[0048] The core of the present invention is to provide a PCR detection composition for Mycobacterium tuberculosis, and the PCR detection composition includes the following primers and probes:

[0049] Primer set for detecting IS6110 sequence:

[0050] IS6110-F5, having the nucleotide sequence shown in SEQ ID NO:1,

[0051] IS6110-R5, having the nucleotide sequence shown in SEQ ID NO:2,

[0052] IS6110-P5, having the nucleotide sequence shown in SEQ ID NO:3,

[0053] Primer set for detecting IS1081 sequence:

[0054] IS1081-F7, having the nucleotide sequence shown in SEQ ID NO:4,

[0055] IS1081-R7, having the nucleotide sequence shown in SEQ ID NO:5,

[0056] IS1081-P7, having the nucleotide sequence shown in SEQ ID NO:6,

[0057] Internal standard primer set for tongue swab detection:

[0058] Actin-F3, having the nucleotide sequence shown in SEQ ID NO:7,

[0059] Actin-R3, having the nucleotide sequence shown in SEQ ID NO:8,

[0060] Actin-P3 has a nucleotide sequence as shown in SEQ ID NO: 9,

[0061] wherein the specific probe is modified with a fluorescent group at the 5'-end and / or a quenching group at the 3'-end.

[0062] Another core of the present invention is to provide a tongue swab PCR detection reagent based on the PCR detection composition of Mycobacterium tuberculosis described in the present invention. The tongue swab PCR detection reagent includes a tongue swab nucleic acid rapid extraction reagent and a Mycobacterium tuberculosis detection reagent. The tongue swab nucleic acid rapid extraction reagent includes: Triton X-100, Tris-HCl, EDTA, Chelex-100, digitonin, and dodecyl dimethyl betaine; the Mycobacterium tuberculosis detection reagent includes: the PCR detection composition of Mycobacterium tuberculosis described in the present invention, PCR buffer, dNTPs, hot start enzyme, UNG enzyme, reverse transcriptase, graphene oxide, and betaine.

[0063] In an alternative embodiment, the tongue swab nucleic acid rapid extraction reagent includes the following components at final concentrations: Triton X-100 1 wt%, Tris-HCl 10 mM, EDTA 1 mM, Chelex-100 0.5 - 2 wt%, digitonin 1 - 3 wt%, and dodecyl dimethyl betaine 1 - 3 wt%, with a pH of 7.0 - 8.0. As a preferred embodiment, the tongue swab nucleic acid rapid extraction reagent includes Chelex-100 1 wt%, digitonin 1.5 wt%, and dodecyl dimethyl betaine 2 wt% at final concentrations. A more preferred embodiment is that the tongue swab nucleic acid rapid extraction reagent includes the following components at final concentrations: Triton X-100 1 wt%, Tris-HCl 10 mM, EDTA 1 mM, Chelex-100 1 wt%, digitonin 1.5 wt%, and dodecyl dimethyl betaine 2 wt%, with a pH of 7.0 - 8.0.

[0064] In an alternative embodiment, the Mycobacterium tuberculosis detection reagent includes the following components at final concentrations: the PCR detection composition of Mycobacterium tuberculosis described in the present invention, 1xPCR buffer, Taq enzyme 6 U, MgCl2 3 mM, graphene oxide 1 - 2 ng / mL, and betaine 0.5 - 1 M.

[0065] In an alternative embodiment, the detection method of the reagent includes the following steps: Mix the tongue swab nucleic acid rapid extraction reagent with a tongue swab sample, heat in a water bath at 95°C for 10 minutes, centrifuge at 12000 r / min for 3 minutes, and take the supernatant to complete the rapid nucleic acid extraction. This detection method can complete the detection of Mycobacterium tuberculosis nucleic acid within 1 hour.

[0066] Preferably, the selected tongue swab sampling site is the root of the tongue, and the sampling time each time is not less than 15 seconds.

[0067] Another core of the present invention is to provide a tongue swab detection kit, which contains the tongue swab PCR detection reagent described in the present invention.

[0068] Design and screening of primers and probes for detecting Mycobacterium tuberculosis in Example 1, Comparative Example 1, and Comparative Example 2

[0069] Example 1

[0070] To ensure the detection of Mycobacterium tuberculosis and avoid false positives or false negatives, the conserved regions of the specific IS1081 sequence and IS6110 sequence of Mycobacterium tuberculosis were selected as targets for detection, and multiple sets of primers were designed for multiple different sites of the IS1081 sequence and IS6110 sequence respectively, and each set of primers was tested and screened. Based on a large number of primer design and screening results, finally, 1 set of the best primers was selected from 10 pairs of primer sets.

[0071] Primer set for detecting the IS6110 sequence: IS6110-F5, having the nucleotide sequence shown in SEQ ID NO:1, IS6110-R5, having the nucleotide sequence shown in SEQ ID NO:2, IS6110-P5, having the nucleotide sequence shown in SEQ ID NO:3.

[0072] Primer set for detecting the IS1081 sequence: IS1081-F7, having the nucleotide sequence shown in SEQ ID NO:4, IS1081-R7, having the nucleotide sequence shown in SEQ ID NO:5, IS1081-P7, having the nucleotide sequence shown in SEQ ID NO:6.

[0073] Due to excessive content, only some results are selected as comparative examples for presentation:

[0074] Comparative Example 1

[0075] Primer set for detecting the IS6110 sequence: IS6110-F2, having the nucleotide sequence shown in SEQ ID NO:10, IS6110-R2, having the nucleotide sequence shown in SEQ ID NO:11, IS6110-P2, having the nucleotide sequence shown in SEQ ID NO:12.

[0076] Primer sets for detecting IS1081 sequence: IS1081-F9, having the nucleotide sequence shown in SEQ ID NO:13; IS1081-R9, having the nucleotide sequence shown in SEQ ID NO:14; IS1081-P9, having the nucleotide sequence shown in SEQ ID NO:15.

[0077] Comparative Example 2

[0078] Primer sets for detecting IS6110 sequence: IS6110-F3, having the nucleotide sequence shown in SEQ ID NO:16; IS6110-R3, having the nucleotide sequence shown in SEQ ID NO:17; IS6110-P3, having the nucleotide sequence shown in SEQ ID NO:18.

[0079] Primer sets for detecting IS1081 sequence: IS1081-F8, having the nucleotide sequence shown in SEQ ID NO:19; IS1081-R8, having the nucleotide sequence shown in SEQ ID NO:20; IS1081-P8, having the nucleotide sequence shown in SEQ ID NO:21.

[0080] Based on the primer sets for detecting IS1081 sequence and IS6110 sequence, an internal reference gene was also set: Actin-F3, having the nucleotide sequence shown in SEQ ID NO:7; Actin-R3, having the nucleotide sequence shown in SEQ ID NO:8; Actin-P3, having the nucleotide sequence shown in SEQ ID NO:9.

[0081] The specific PCR detection compositions described in Example 1 and Comparative Examples 1 and 2 are shown in Table 1. The specific steps for performing fluorescence quantitative PCR detection of samples using them are as follows:

[0082] Table 1 Primers and Probes Described in Example 1 and Comparative Examples 1 and 2

[0083]

[0084]

[0085] 1. Sample acquisition and processing

[0086] An inactivated bacterial solution of the standard strain H37Rv and a human oral swab were obtained. After extraction with the Novoprotein VAMNE Magnetic Pathogen DNA Kit, the nucleic acid of the sample was diluted with a 2 ng / mL human oral swab DNA solution and stored for subsequent steps.

[0087] Construct a plasmid containing the IS6110 and IS1081 gene fragments and dilute it with a 2 ng / mL human oral swab DNA solution.

[0088] 2. Reaction system and reaction program

[0089] Perform fluorescence quantitative PCR detection using the PCR reaction system shown in Table 2.

[0090] Table 2 PCR reaction system

[0091] Material Final concentration Dosage in 50 μL system (μL) 10×qPCR buffer (containing UNG enzyme) 1× 5 Taq enzyme (5 U) 6U 1.2 <![CDATA[MgCl2(200mM)]]> 3 mM 0.75 IS6110 forward primer (100 mM) 0.3 μM 0.15 IS6110 reverse primer (100 mM) 0.3 μM 0.15 IS6110 fluorescent probe (100 mM) 0.15 μM 0.075 IS1081 forward primer (100 mM) 0.5 μM 0.25 IS1081 reverse primer (100 mM) 0.5 μM 0.25 IS1081 fluorescent probe (100 mM) 0.25 μM 0.125 Internal standard forward primer (100 mM) 0.1 μM 0.05 Internal standard reverse primer (100 mM) 0.1 μM 0.05 Internal standard fluorescent probe (100 mM) 0.05 μM 0.025 <![CDATA[H2O]]> 16.925 Template DNA 25 Total 50 μL

[0092] The above reaction solution can be adapted to a variety of fluorescence quantitative PCR instruments. In this example, taking the Hongshi SLAN96 as an example, the reaction program is: 25°C for 5 minutes to activate the UNG enzyme; 95°C for 30 s for pre-denaturation; 95°C for 5 s for denaturation, 65°C for 20 s for annealing and collecting fluorescence signals, for 45 cycles. The heating and cooling rate is set at 1.6°C / s.

[0093] Set a positive control and a negative control for each group of reactions.

[0094] 3. Interpretation of reaction results

[0095] The instrument collects fluorescence-labeled probe signals of different wavelengths, and after software processing, amplification curves of two fluorescence channels, FAM and CY5, are presented. The positive and negative of Mycobacterium tuberculosis are judged according to different fluorescence signals and CT values. Among them, the positive control is positive, the negative control is negative, and the internal standard amplification is normal, indicating that the results are valid.

[0096] The detection results are as Figure 1 shown. Among them, both the test sample and the positive control show typical amplification curves, and the CT value is below 40, the internal standard amplification is normal, and there is no non-specific amplification in the negative control, indicating that the present invention has good Mycobacterium tuberculosis detection ability. Compared with other primer sets, the primer set selected in the present invention (9 nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9) has the advantages of high sensitivity and fast amplification speed.

[0097] Example 2 Specificity experiment of the primer and probe combination for detecting Mycobacterium tuberculosis

[0098] The present invention relates to a variety of primer-probes. When forming a multiplex reaction system, non-specific amplification or cross-reaction phenomena may occur. To verify the specificity of the present invention, nucleic acids of Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli, Staphylococcus epidermidis, Staphylococcus aureus, Nocardia, Candida albicans, human influenza virus, and human parainfluenza virus were collected from the perspectives of gene sequence homology, clinical symptom similarity, and proximity of infection sites, as well as nucleic acids of the following pathogens: Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium gordonae, Mycobacterium xenopi, Mycobacterium avium, Mycobacterium scrofulaceum, Mycobacterium szulgai, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium phlei. Together with the control group of Mycobacterium tuberculosis, a total of 24 pathogen standard strains were used for nucleic acid extraction with the Novizan VAMNE Magnetic Pathogen DNA Kit. After being verified by sequencing, digital PCR was used for quantification. It was diluted to 2000 copies / μL with a 2 ng / mL human oral swab DNA solution for standby. Referring to Table 2 of Example 1, the tongue swab PCR detection reagent was prepared, and the primer set was 9 nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9. 25 μL of the above pathogen DNA template was added to 25 μL of the tongue swab PCR detection reagent for specificity testing.

[0099] Table 3 Specificity test results

[0100]

[0101]

[0102] The specificity test results are shown in Table 3 and Figure 2 As shown, except for Mycobacterium tuberculosis being positive, the remaining 23 pathogens were all negative. After 20 replicates of testing for each nucleic acid, the specificity was found to be 100%, indicating good specificity.

[0103] Example 3 Sensitivity test and reaction system optimization

[0104] An inactivated bacterial solution with a concentration of 10 8 CFU / mL of the standard strain H37Rv was obtained. After extraction with the Novizan VAMNE Magnetic Pathogen DNA Kit, it was diluted to 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2CFU / mL, 10 CFU / mL, and store for subsequent steps.

[0105] Refer to Table 2 of Example 1 to prepare the tongue swab PCR detection reagent. The primer set is 9 nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:9. Add 25 μL of H37Rv DNA templates with concentrations of 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, and 10 CFU / mL to 25 μL of the tongue swab PCR detection reagent respectively.

[0106] Experimental group 1: Add graphene oxide to the reaction system so that the final concentration of graphene oxide in the reaction system is 1.6 ng / mL.

[0107] Experimental group 2: Add betaine to the reaction system so that the final concentration of betaine in the reaction system is 0.5 M.

[0108] Experimental group 3: Add graphene oxide and betaine to the reaction system so that the final concentration of graphene oxide in the reaction system is 1.6 ng / mL and the final concentration of betaine in the reaction system is 0.5 M.

[0109] Experimental group 4: Add trehalose to the reaction system so that the final concentration of trehalose in the reaction system is 0.2 M.

[0110] Control group 1: Do not add extra.

[0111] Use the method described in Example 1 to detect the above-diluted samples. Each group of samples is repeated 20 times. Statistically analyze the results with CT values below 40 and calculate the detection rate. The results are shown in Table 4.

[0112] Table 4: Sensitivity test results

[0113] Group <![CDATA[10 5 CFU / mL]]> <![CDATA[10 4 CFU / mL]]> <![CDATA[10 3 CFU / mL]]> <![CDATA[10 2 CFU / mL]]> 10 CFU / mL NTC Experimental group 1 100％ 100％ 100％ 70％ 0％ 0％ Experimental group 2 100％ 100％ 100％ 90％ 65％ 0％ Experimental group 3 100％ 100％ 100％ 100％ 95％ 0％ Experimental group 4 100％ 100％ 100％ 90％ 65％ 0％ Control group 1 100％ 100％ 100％ 85％ 45％ 0％

[0114] As can be seen from the results in Table 4, compared with other groups, the sensitivity result of Experimental group 3 is better. Adding graphene oxide and betaine to the reaction system has a higher PCR enhancement effect than adding graphene oxide, betaine, and trehalose alone. Adding an appropriate amount of graphene oxide to the reaction system, graphene oxide forms a primer-graphene oxide complex by adsorbing free primers, improving the capture ability for the template and enhancing the detection rate of low-concentration templates. At the same time, adding an appropriate amount of betaine is beneficial to reducing the formation of secondary structures to improve DNA amplification.

[0115] Optimization of the Rapid Nucleic Acid Extraction Reagent for Tongue Swabs and Comparative Experiments with Column Extraction and Magnetic Bead Extraction Methods

[0116] Obtain an inactivated bacterial solution with a concentration of 10 8 CFU / mL of the standard strain H37Rv, and dilute it to 10 6 CFU / mL, 10 5 CFU / mL, 10 4 CFU / mL, 10 3 CFU / mL, 10 2 CFU / mL, 10 CFU / mL, 1 CFU / mL.

[0117] Refer to Table 2 in Example 1 to prepare the PCR detection reagent for tongue swabs, where the primer set is 9 nucleotide sequences shown in SEQ ID NO:1 to SEQ ID NO:9.

[0118] Experimental groups 5, 6, 7 and control groups 2, 3, 4: Prepare the rapid nucleic acid extraction reagent for tongue swabs according to Table 5 and adjust the pH to 7.0 - 8.0. Take 100 μL of the rapid nucleic acid extraction reagent for tongue swabs, add 100 μL of Mycobacterium tuberculosis bacterial solution to it, heat in a water bath at 95°C for 10 minutes, centrifuge at 12000 r / min for 3 minutes, take 25 μL of the supernatant and add it to 25 μL of the PCR detection reagent for tongue swabs for detection.

[0119] Table 5: Components of the Rapid Nucleic Acid Extraction Reagent for Tongue Swabs (Final Concentration)

[0120]

[0121] Control group 5: The experimental method is the same as that of experimental group 5, the difference is that after adding the bacterial solution to the rapid nucleic acid extraction reagent for tongue swabs, add 20 μL of proteinase K, heat in a water bath at 50°C for 15 minutes, heat in a water bath at 95°C for 10 minutes, centrifuge at 12000 r / min for 3 minutes, take 25 μL of the supernatant and add it to 25 μL of the PCR detection reagent for tongue swabs for detection.

[0122] Control group 6: The experimental method is the same as that of experimental group 5, the difference is that after adding the bacterial solution, shake it with a vortex mixer for 2 minutes, centrifuge at 12000 r / min for 3 minutes, take 25 μL of the supernatant and add it to 25 μL of the PCR detection reagent for tongue swabs for detection.

[0123] Control group 7: The column extraction method refers to the extraction using the Novoprotein FastPure Cell / Tissue DNA Isolation mini Kit. Add 100 μL of Mycobacterium tuberculosis bacterial solution to it, elute it with 100 μL of TE, and then take 25 μL and add it to 25 μL of the PCR detection reagent for tongue swabs for detection.

[0124] Control Group 8: The magnetic bead extraction method was referred to the extraction method of Novoprotein VAMNE Magnetic Pathogen DNA Kit. 100 μL of Mycobacterium tuberculosis bacterial solution was added to it. After elution with 100 μL of TE, 25 μL was taken and added to 25 μL of the tongue swab PCR detection reagent for detection.

[0125] The above detection was repeated 20 times for each group of samples. The results with a CT value of less than 40 were statistically analyzed, and the detection rates were calculated. The statistical results are shown in Table 6. Among them, the amplification results of Experimental Group 5 are as Figure 3 shown, the amplification results of Control Group 7 are as Figure 4 shown, and the amplification results of Control Group 8 are as Figure 5 shown.

[0126] Table 6: Statistical Results of Detection Rates of Different Sample Processing Methods

[0127] Group <![CDATA[10 5 CFU / mL]]> <![CDATA[10 4 CFU / mL]]> <![CDATA[10 3 CFU / mL]]> <![CDATA[10 2 CFU / mL]]> 10 CFU / mL NTC Experimental group 5 100％ 100％ 100％ 100％ 95％ 0％ Experimental group 6 100％ 100％ 100％ 100％ 85％ 0％ Experimental group 7 100％ 100％ 100％ 100％ 80％ 0％ Experimental group 8 100％ 100％ 100％ 100％ 75％ 0％ Experimental group 9 100％ 100％ 100％ 90％ 85％ 0％ Experimental group 10 100％ 100％ 100％ 90％ 80％ 0％ Experimental group 11 100％ 100％ 100％ 95％ 85％ 0％ Control group 2 100％ 100％ 90％ 65％ 25％ 0％ Control group 3 100％ 100％ 100％ 85％ 45％ 0％ Control group 4 100％ 100％ 100％ 75％ 25％ 0％ Control group 5 100％ 100％ 100％ 100％ 95％ 5％ Control group 6 100％ 100％ 100％ 95％ 65％ 0％ Control group 7 100％ 100％ 100％ 95％ 25％ 10％ Control group 8 100％ 100％ 100％ 95％ 90％ 5％

[0128] It can be seen from Experimental Groups 5, 6, 9 and Control Group 2 in Table 6 that adding Chelex-100 to the tongue swab nucleic acid rapid extraction reagent can improve the detection rate of low-concentration samples, and the optimal addition amount is 1 wt%. It can be seen from Experimental Groups 5, 7, 10 and Control Group 3 in Table 6 that adding digitonin to the tongue swab nucleic acid rapid extraction reagent can improve the detection rate of low-concentration samples, and the optimal addition amount is 1.5 wt%. It can be seen from Experimental Groups 5, 8, 11 and Control Group 4 in Table 6 that adding dodecyldimethylbetaine to the tongue swab nucleic acid rapid extraction reagent can improve the detection rate of low-concentration samples, and the optimal addition amount is 2 wt%. Control Group 5 shows that although adding proteinase K to the lysis solution can improve the detection rate of low-concentration samples, opening the lid to add liquid will increase the operation process and bring the possibility of sample contamination. In summary, the optimal combination in the tongue swab nucleic acid rapid extraction reagent is Triton X-100 1 wt%, Tris-HCl 10 mM, EDTA 1 mM, Chelex-100 1 wt%, digitonin 1.5 wt% and dodecyldimethylbetaine 2 wt%, and the pH is 7.0 - 8.0.

[0129] It can be seen from Table 6 and Figures 3 - 5 that the efficiency of the tongue swab nucleic acid rapid extraction reagent in lysing and releasing TB nucleic acid is comparable to that of the magnetic bead method and is superior to the column extraction method. Compared with the column extraction method and the magnetic bead method, the tongue swab nucleic acid rapid extraction reagent provided by the present invention has fewer steps in the lysis process, reduces the chance of contamination, and reduces the loss of nucleic acid.

[0130] Effect of Sample Sampling Site and Sampling Time on Detection Results in Example 5

[0131] Experimental samples: Samples of patients positive for Mycobacterium tuberculosis provided by the hospital (40 cases), throat swab samples, tongue swab samples from different parts of the tongue (root of the tongue, middle of the tongue, tip of the tongue), and sputum samples collected from the same patients. Among them, the sputum specimens were verified as positive patients by sputum culture. See the schematic diagram of different sampling sites in Figure 6 。

[0132] Experimental group 12: Collect the root of the tongue of the patient. The sampling method is to gently scrape the surface of the root of the tongue with a tongue swab for 15 seconds. After extracting the above tongue swab sample according to the experimental group 5 in Example 4, take 25 μL of the supernatant and detect it according to the experimental group 3 in Example 3.

[0133] Control group 9: The experimental method is the same as that of experimental group 12, except that the sampling site is the middle of the tongue.

[0134] Control group 10: The experimental method is the same as that of experimental group 12, except that the sampling site is the tip of the tongue.

[0135] Control group 11: The experimental method is the same as that of experimental group 12, except that the sampling site is the throat swab.

[0136] Control group 12: The experimental method is the same as that of experimental group 12, except that the sampling duration is 30 seconds.

[0137] The above experimental groups and control groups were all detected according to the reaction procedure in Example 1, and their positive detection rates (the proportion of positive judgment results in the total number of test samples) and missed diagnosis rates (the proportion of the number of samples with inconsistent judgment results and sputum culture in the number of samples positive for sputum culture) were statistically analyzed. The test results of different sampling sites and sampling methods are shown in Table 8.

[0138] Table 7: Statistical results of test results of different sampling sites and sampling methods

[0139] Group Positive detection rate Missed diagnosis rate Experimental group 12 85％ 15％ Control group 9 65％ 35％ Control group 10 55％ 45％ Control group 11 70％ 30％ Control group 12 80％ 20％

[0140] As can be seen from Table 7, the positive detection rate of the tongue swab with the sampling site at the root of the tongue is higher than that of the throat swab and the tongue swabs at the middle and tip of the tongue. This may be related to the load of Mycobacterium tuberculosis in different parts of the oral cavity. The positive detection rate of the tongue swab with a sampling duration of 15 seconds is higher than that of the tongue swab with a sampling duration of 30 seconds. It is speculated that when the sampling duration is too long, the inhibitory substances such as saliva secreted by the patient's oral cavity increase, resulting in missed detection of the sample.

[0141] Application of the tongue swab PCR detection reagent in Example 6

[0142] A tongue swab PCR detection reagent consists of a tongue swab nucleic acid rapid extraction reagent and a Mycobacterium tuberculosis detection reagent.

[0143] The rapid nucleic acid extraction reagent for tongue swabs consists of components with the following final concentrations: Triton X-100 1 wt%, Tris-HCl 10 mM, EDTA 1 mM, Chelex-100 1 wt%, digitonin 1.5 wt%, and dodecyldimethylbetaine 2 wt%, with a pH of 7.0 - 8.0.

[0144] The Mycobacterium tuberculosis detection reagent consists of components with the following final concentrations: the PCR detection composition for Mycobacterium tuberculosis of the present invention (the primer set is 9 nucleotide sequences shown in SEQ ID NO: 1 to SEQ ID NO: 9), 1xPCR buffer, Taq enzyme 6 U, MgCl2 3 mM, graphene oxide 1.6 ng / mL, and betaine 0.5 M.

[0145] Experimental samples: Samples of suspected Mycobacterium tuberculosis patients provided by the hospital (80 cases), tongue swab samples and sputum samples were taken from the same patients. Among them, the sputum specimens were detected by sputum culture, and the sputum culture results were used as the gold standard to judge the detection accuracy of tongue swabs.

[0146] Experimental group 13: Add 200 μL of the above-mentioned rapid nucleic acid extraction reagent for tongue swabs to the tongue swab, heat in a water bath at 95°C for 10 minutes, centrifuge at 12,000 r / min for 3 minutes, take 25 μL of the supernatant, and add 25 μL of the Mycobacterium tuberculosis detection reagent for detection.

[0147] Control group 13: Take 400 μL of normal saline, add it to the tongue swab, shake with a vortex mixer for 2 minutes, take 200 μL of the supernatant, extract nucleic acid using the Novizan VAMNE Magnetic Pathogen DNA Kit kit, elute with 100 μL of TE, take 25 μL, and add 25 μL of the Mycobacterium tuberculosis detection reagent for detection.

[0148] The above reaction solution can be adapted to a variety of fluorescence quantitative PCR instruments. Taking the Hongshi SLAN96 as an example in this embodiment, the reaction program is: 25°C for 5 minutes to activate the UNG enzyme; 95°C for 30 s for pre-denaturation; 95°C for 5 s for denaturation, 65°C for 20 s for annealing and collecting fluorescence signals, for 45 cycles. The heating and cooling rate is set to 1.6°C / s.

[0149] Statistical analysis was performed on its positive detection rate (the proportion of positive judgment results in the total number of test samples), and the missed diagnosis rate (the proportion of the number of samples with inconsistent judgment results and sputum culture in the samples with positive sputum culture). The test results are shown in Table 8.

[0150] Table 8: Statistical analysis of test results

[0151]

[0152]

[0153] As can be seen from Table 8, the rapid extraction Mycobacterium tuberculosis detection reagent for tongue swabs provided by the present invention is applicable to the detection of tongue swab samples.

[0154] In summary, the rapid extraction Mycobacterium tuberculosis detection reagent for tongue swabs provided by the present invention adds Chelex-100, digitonin, and dodecyldimethylbetaine to the rapid extraction component. The combined use of the three is beneficial to the lysis of Mycobacterium tuberculosis. The whole process has fewer steps, reduces the chance of contamination, reduces the loss of nucleic acid, and improves the quality of tongue swab nucleic acid samples. By adding graphene oxide and betaine to the reaction system, the capture ability of the template is improved, and the detection rate of low-concentration templates is increased. At the same time, the root of the tongue with a higher bacterial load is collected, and the sampling duration is optimized to avoid partial missed detection of Mycobacterium tuberculosis, thus solving the problem of low sensitivity of tongue swabs.

[0155] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A tongue swab PCR detection reagent, characterized in that: The tongue swab PCR detection reagent includes a tongue swab nucleic acid rapid extraction reagent and a Mycobacterium tuberculosis detection reagent, wherein The tongue swab nucleic acid rapid extraction reagent includes the following components at final concentrations: Triton X-100 1wt%, Tris-HCl 10mM, EDTA 1mM, Chelex-100 1wt%, digitonin 1.5wt% and dodecyl dimethyl betaine 2wt%, pH 7.0-8.0; The Mycobacterium tuberculosis detection reagent includes the following components at final concentrations: a PCR detection composition for Mycobacterium tuberculosis, 1xPCR buffer, 6U of Taq enzyme, 3mM MgCl2, 1-2ng / mL of graphene oxide, and 0.5-1M of betaine; the Mycobacterium tuberculosis PCR detection composition includes the following primers and probes: Primer set for detecting IS6110 sequence: IS6110-F5, having the nucleotide sequence shown in SEQ ID NO: 1, IS6110-R5, having the nucleotide sequence shown in SEQ ID NO:2, IS6110-P5, having the nucleotide sequence shown in SEQ ID NO:3, Primer set for detecting IS1081 sequence: IS1081-F7, having the nucleotide sequence shown in SEQ ID NO:4, IS1081-R7, having the nucleotide sequence shown in SEQ ID NO:5, IS1081-P7, having the nucleotide sequence shown in SEQ ID NO:6, Internal standard primer set for tongue swab test: Actin-F3, having the nucleotide sequence shown in SEQ ID NO:7, Actin-R3, having the nucleotide sequence shown in SEQ ID NO:8, Actin-P3, having the nucleotide sequence shown in SEQ ID NO:9, The specific probe is modified with a fluorescent group at the 5' end and / or a quenching group at the 3' end, wherein the fluorescent group is selected from any one of FAM, VIC, ROX, and CY5; the quenching group is selected from BHQ1 or BHQ2, the probes IS6110-P5 and IS1081-P7 are labeled with FAM, and the probe Actin-P3 is labeled with CY5.

2. The tongue swab PCR detection reagent according to claim 1, characterized in that: The detection method of the reagent comprises the following steps: mixing the tongue swab nucleic acid rapid extraction reagent with the tongue swab sample, heating in a 95°C water bath for 10 minutes, centrifuging at 12000r / min for 3 minutes, and taking the supernatant to complete the rapid nucleic acid extraction.

3. The tongue swab PCR detection reagent according to claim 2, characterized in that: The selected tongue swab sampling site is the root of the tongue, and each sampling time is no less than 15 seconds.

4. A tongue swab detection kit, comprising the tongue swab PCR detection reagent according to claim 1.

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