Mycobacterium tuberculosis complex and rifampicin drug-resistant mutation detection kit and method

Abstract

The invention discloses a detection kit and method for Mycobacterium tuberculosis complex and rifampicin drug-resistant mutation, and belongs to the technical field of biologication.A Taqman probe and a molecular beacon are combined, a fluorescent signal is generated based on a Taq enzyme hydrolysis fluorescent probe, the fluorescent signal is accumulated, and the molecular beacon is used for detecting the drug-resistant mutation of the Mycobacterium tuberculosis complex and the rifampicin drug-resistant mutation of the Mycobacterium tuberculosis complex and the rifampicin drug-resistant mutation. According to two technical principles that a molecular beacon and a fluorescent product are hybridized to generate a fluorescent signal, fluorescent PCR amplification is adopted for detection of one target spot in a monochromatic fluorescent channel, and a melting curve mode of a probe and a target gene is adopted for detection of the other target spot. Therefore, two target spots can be detected and analyzed in a monochromatic fluorescence channel at the same time. Single-tube multiple detection is achieved, and mycobacterium tuberculosis complex group detection and mycobacterium tuberculosis rifampicin drug-resistant mutation site detection can be achieved at the same time.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a detection kit and method for Mycobacterium tuberculosis complex and rifampicin resistance mutation. Background Art

[0002] Tuberculosis (TB) is a global chronic respiratory infectious disease. Although my country has made great progress in the prevention and control of tuberculosis, the prevalence of tuberculosis is still serious. Tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis and can invade various organs of the body, but pulmonary tuberculosis is the most common, accounting for 80%-90% of the total number of tuberculosis in various organs, and those who excrete bacteria in sputum are called infectious pulmonary tuberculosis. It is mainly transmitted by inhaling infectious bacteria in the air after sputum excreted by infectious pulmonary tuberculosis patients. Susceptible people are mostly groups with altered or low immune function. Drug resistance is the main obstacle to the treatment of tuberculosis and poses a challenge to global public health and therapeutics. Although anti-tuberculosis drugs are effective, drug-resistant isolates of Mycobacterium tuberculosis have also appeared. According to the World Health Organization, there were about 410,000 new cases of rifampicin-resistant tuberculosis (RR-TB) in the world in 2002. Treatment delay in tuberculosis patients is an important factor hindering tuberculosis control. Treatment delay in RR-TB patients can lead to disease progression, increased risk of death, and further spread of drug-resistant tuberculosis. Therefore, rapid and accurate identification of Mycobacterium tuberculosis complex and screening for rifampicin resistance are of great significance for the early diagnosis and treatment of tuberculosis and the prevention and control of tuberculosis.

[0003] The main target of tuberculosis diagnosis is the unique conservative housekeeping gene in the Mycobacterium tuberculosis genome. Commonly used target genes include IS6110, 16S ribosomal RNA (16SrRNA), gyrB and rpoB genes, among which 16srRNA, gyrB and rpoB genes are single-copy genes in the MTB genome, and IS6110 is a multi-copy gene. Generally, the sensitivity of the method using single-copy genes as the detection target is lower than that of the method using multi-copy genes as the detection target. Moreover, IS6110 is a transposable gene of the Mycobacterium tuberculosis complex, a common insertion sequence, about 1361bp long, with different numbers of copies in the Mycobacterium tuberculosis complex but not in other species of the genus Mycobacterium. IS1081 also has multiple copies in the Mycobacterium tuberculosis complex. Drug resistance of Mycobacterium tuberculosis is mainly caused by random mutations of drug target genes in the genome. The target protein of the first-line anti-tuberculosis drug rifampicin in bacteria is the β subunit of RNA polymerase. Rifampicin binds to the β subunit of RNA polymerase, hindering mRNA synthesis, inhibiting the transcription process, and blocking bacterial protein synthesis, achieving an antibacterial effect. Studies have shown that rifampicin resistance in Mycobacterium tuberculosis is usually caused by mutations in the gene encoding the β subunit of RNA polymerase, rpoB.

[0004] The following related technologies exist in the prior art: 1. A single tube only detects Mycobacterium tuberculosis complex, 2. A single tube only detects Mycobacterium tuberculosis rifampicin resistance. These two methods need to be used in combination to detect both Mycobacterium tuberculosis complex and Mycobacterium tuberculosis rifampicin resistance, which increases the number of tests and the cost of testing; 3. A single multi-tube simultaneous detection of Mycobacterium tuberculosis complex and its rifampicin resistance. Currently, the PCR detection kit using this method can only detect a single target in a single channel. The existing commonly used fluorescence quantitative instrument has 4 detection channels, and the number of targets detected in a single tube is generally 4. In practice, this limits the single tube to detect five or six targets at the same time; detecting five or six targets at the same time requires multi-tube multiplex testing. Summary of the invention

[0005] In response to the technical problem raised in the above background technology of how to realize the detection of Mycobacterium tuberculosis complex and multiple detection of rifampicin-resistant mutation sites of Mycobacterium tuberculosis through a single tube and one channel, the present invention proposes a detection kit and method for Mycobacterium tuberculosis complex and rifampicin-resistant mutation.

[0006] In the first aspect, an embodiment of the present invention provides a nucleic acid composition, which includes: a first primer pair with a nucleotide sequence as shown in SEQ ID No.1-2; a second primer pair with a nucleotide sequence as shown in SEQ ID No.3-4; and a third primer pair with a nucleotide sequence as shown in SEQ ID No.5-6.

[0007] The nucleic acid composition further comprises probes with sequences such as SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12.

[0008] In a second aspect, an embodiment of the present invention provides a detection reagent, which includes the nucleic acid composition described in the aforementioned embodiment.

[0009] In a third aspect, an embodiment of the present invention provides a detection kit, which includes the nucleic acid composition described in the preceding embodiment or the detection reagent described in the preceding embodiment.

[0010] In a fourth aspect, embodiments of the present invention provide use of the nucleic acid composition described in the preceding embodiments in the preparation of a detection reagent or kit for Mycobacterium tuberculosis and / or a rifampicin resistance gene mutation of Mycobacterium tuberculosis.

[0011] In the above technical solution, the samples to be tested include different samples such as sputum, alveolar lavage fluid, etc.

[0012] In a fifth aspect, an embodiment of the present invention provides a method for detecting Mycobacterium tuberculosis and / or its rifampicin gene mutation, which comprises: performing PCR detection on the sample using the nucleic acid composition described in the preceding embodiment, or the detection reagent described in the preceding embodiment, or the detection kit described in the preceding embodiment; the detection method is not directly intended for the diagnosis or treatment of the disease.

[0013] In the above technical solution, the detection method also includes a method for determining whether the Mycobacterium tuberculosis complex is negative or positive and whether the rifampicin gene mutation is sensitive or resistant based on the simultaneous detection of two target points in a monochromatic fluorescence channel.

[0014] Beneficial Effects

[0015] The present invention adopts a combination of Taqman probe and molecular beacon, based on two technical principles that Taq enzyme hydrolyzes fluorescent probe to generate fluorescent signal, and the fluorescent signal is cumulative, and molecular beacon hybridizes with fluorescent product to generate fluorescent signal, and adopts fluorescent PCR amplification for the detection of one target point in the monochrome fluorescent channel, and adopts the melting curve of probe and target gene for the detection of another target point, thereby realizing the detection and analysis of two target points in the monochrome fluorescent channel at the same time. It realizes single-tube multiple detection, and can realize the detection of Mycobacterium tuberculosis complex group and the detection of Mycobacterium tuberculosis rifampicin resistance mutation site at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 This is the test result of the reagent of the present invention on the national reference plate of tuberculosis (ROX channel) (a).

[0018] Figure 2 This is the test result of the reagent of the present invention on the national reference plate of tuberculosis (ROX channel) (ii).

[0019] Figure 3 The results of the test of the national reference plate for tuberculosis by the reagent of the present invention (CY5 channel) (a).

[0020] Figure 4 This is the test result of the reagent of the present invention on the national reference plate of tuberculosis (CY5 channel) (ii).

[0021] Figure 5 It is the test result (VIC channel) of the national reference plate of tuberculosis by the reagent of the present invention.

[0022] Figure 6 This is the test result of the reagent of the present invention on the national reference plate of tuberculosis (FAM channel).

[0023] Figure 7 It is the test result (ROX channel) of the reagent of the present invention to the sensitive reference material of the national reference material plate of rifampicin resistance.

[0024] Figure 8 It is the test result (FAM channel) of the reagent of the present invention to the sensitive reference material of the national reference material plate of rifampicin resistance.

[0025] Fig. 9 It is the test result (VIC channel) of the reagent of the present invention to the sensitive reference material of the national reference material plate of rifampicin resistance.

[0026] Fig.10 It is the test result of the reagent of the present invention on the sensitive reference material of the national reference material plate of rifampicin resistance (CY5 channel).

[0027] Fig.11 It is the test result (ROX channel) of the reagent of the present invention on the rifampicin-resistant national reference plate.

[0028] Fig.12 It is the test result (FAM channel) of the reagent of the present invention on the rifampicin-resistant national reference plate.

[0029] Fig.13 It is the test result of the reagent of the present invention on the rifampicin resistance national reference plate resistance reference (VIC channel).

[0030] Fig.14 It is the test result of the reagent of the present invention on the rifampicin-resistant national reference plate (CY5 channel). DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0032] Example 1 A kit for detecting Mycobacterium tuberculosis complex and rifampicin resistance mutation

[0033] In this embodiment, a pair of primers and a probe (Taqman probe) are designed for each of the IS6110 and IS1081 genes unique to the Mycobacterium tuberculosis complex, which can detect the Mycobacterium tuberculosis complex, with better sensitivity than the single gene IS6110 detection, and higher sensitivity and accuracy; a pair of primers and 4 probes (molecular beacons) are designed for the rpoB resistance-determining region, which can detect 21 common mutation sites of rifampicin resistance. An internal control (internal standard gene) is designed, and a pair of primers and a probe (Taqman probe) are designed for the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in sputum samples to monitor the extraction process and PCR amplification process of the sample to be tested to avoid false negative results.

[0034] As a further improvement to this embodiment, the ratio of upstream and downstream primers of the rpoB target can be further increased (asymmetric PCR), thereby increasing the yield of the target fragment complementary to the probe and amplifying the signal of the melting curve.

[0035] As a further improvement of this embodiment, the fluorescent group includes but is not limited to various commonly used fluorescent markers, such as ALEX-350, AlexaFluor 488, CY3, FAM, VIC, TET, CALGold540, JOE, HEX, CALFluorOrange560, TAMRA, CALFluorRed590, ROX, CALFluorRed610, TexasRed, CALFluorRed635, Quasar670, CY5, CY5.5, LC RED640, Quasar705, etc.

[0036] As a further improvement to this embodiment, the quenching group includes but is not limited to various commonly used quenching agents, such as TAMRA, DABCYL, BHQ-1, BHQ-2, BHQ-3, Eclipse, etc.

[0037] Primer combinations:

[0038] The upstream primer of IS6110 target site is 5'-GAAGGCGTACTCGACCTGAA-3' (as shown in SEQ ID NO.1);

[0039] The primer downstream of the IS6110 target site is 5'-TCTCGGCTAGTGCATTGTCATAG-3' (as shown in SEQ ID NO. 2);

[0040] The primer upstream of the IS1081 target site is 5'-TCGAGGTCCTCACCCGAG-3' (as shown in SEQ ID NO. 3);

[0041] The primer downstream of the IS1081 target site was 5'-GTGGCAGTCTAGGTGGTCAG-3' (as shown in SEQ ID NO. 4);

[0042] The upstream primer of the rpoB target site is 5'-TCCGGGAGCGGATGACCACCC-3' (as shown in SEQ ID NO.5);

[0043] The downstream primer of the rpoB target site is 5'-CCTTGCGGTACGGCGTTTCGAT-3' (as shown in SEQ ID NO.6);

[0044] Probe combination:

[0045] The IS6110 target probe is 5'-CTTCCGACCGCTCCGACCGACG-3' (as shown in SEQ ID NO. 7);

[0046] The IS1081 target probe is 5'-CCGAGCAGCACTGACCAGCACCGAAG-3' (as shown in SEQ ID NO. 8);

[0047] rpoB target probe 1 is:

[0048] 5'-cgcgggCACCAGCCAGCTGAGCCAATTCATGcccgcg-3' (as shown in SEQ ID NO.9);

[0049] rpoB target probe 2 is:

[0050] 5'-cgcgcgGACCAGAACAACCCGCTGTCGGGcgcgcg-3' (as shown in SEQ ID NO.10);

[0051] rpoB target probe 3 is:

[0052] 5'-cgcgcgGGGTTGACCCACAAGCGCCGACTGTCGcgcgcg-3' (as shown in SEQ ID NO. 11);

[0053] rpoB target probe 4 is:

[0054] 5'-cgcgccACCCACAAGCGCCGACTGTCGGCGCTGggcgcg-3' (as shown in SEQ ID NO. 12);

[0055] The above-mentioned internal quality control (Internal Control) includes one upstream primer and one downstream primer designed for internal quality control, and one corresponding probe, and the sequences are as follows.

[0056] The internal control upstream primer is 5′-TGCCAACGTGTCAGCGGTGGAC-3′ (as shown in SEQ ID NO. 13);

[0057] The internal control downstream primer is 5'-CATGGTTCTTCCACCACTTCGTCCGC-3' (as shown in SEQ ID NO. 14);

[0058] The internal control probe is 5′-GGTAGGACCCGATGTGACTCC-3′ (as shown in SEQ ID NO. 15);

[0059] The composition of a detection kit for Mycobacterium tuberculosis complex and rifampicin resistance mutation in this embodiment is shown in Table 1 below.

[0060] Table 1 Kit composition

[0061]

[0062]

[0063] Example 2 A method for detecting Mycobacterium tuberculosis complex and rifampicin resistance mutations

[0064] The detection kit used in the detection method of this embodiment is as described in Example 1. When the kit is used, the PCR reaction conditions include:

[0065] The first stage: 50°C, 2 min; 1 cycle;

[0066] The second stage: 95°C, 10 min; 1 cycle;

[0067] The third stage: 95°C, 40 s to collect fluorescence; 60°C, 40 s; 72°C, 40 s; 45 cycles;

[0068] The fourth stage: 95°C, 2 min, 40°C, 2 min; 1 cycle;

[0069] The fifth stage: 40-85℃, continuously collect fluorescence signals every 0.04℃ / s, and use FAM, VIC, ROX, and CY5 as fluorescence channels.

[0070] The interpretation criteria of the detection method are as follows:

[0071] 1. The target detection signal of Mycobacterium tuberculosis complex is the amplification curve of ROX channel;

[0072] The target detection signals of Mycobacterium tuberculosis combined with rifampicin resistance are the melting curves of FAM, HEX, ROX, and CY5 channels respectively; the internal standard detection signal is the amplification curve of CY5 channel;

[0073] 2. Determination of positive and negative results and rifampicin resistance:

[0074] (1) First analyze whether the internal standard detects the amplification curve in the CY5 channel and CT≤37, indicating that this test is valid and subsequent analysis can be continued:

[0075] (2) If a typical S-shaped amplification curve is detected in the ROX channel and Ct≤37, it means that the Mycobacterium tuberculosis complex test result is positive; rifampicin resistance analysis can be performed;

[0076] (3) The judgment values ​​of positive and negative quality control products are shown in Table 2 below:

[0077] Table 2 Judgment of positive and negative quality control products

[0078]

[0079] The interpretation of the test results is as follows: By comparing the difference in the melting point (Tm value) of the melting curve between the tested sample and the positive control, it is determined whether the sample has mutated. When the melting point of the sample in the four channels is consistent with the melting point of the positive control (the error does not exceed 1.5°C), it is determined to be wild type, and the Mycobacterium tuberculosis in the sample is sensitive to rifampicin; when the absolute value of the difference between the melting point of any channel of the sample in the four channels and the melting point of the positive control is 2.0°C or above, it is determined to be mutant, and the Mycobacterium tuberculosis in the sample is resistant to rifampicin;

[0080] Interpretation of heterozygous sample results: When a double peak or fusion peak appears in the melting curve of any of the four channels, it is a heterozygous sample, which is divided into three situations: 1. When a double peak appears, it is a heterozygous sample; 2. When there is a fusion peak, it is consistent with the melting point of the positive control, but the peak shape is quite different from that of the positive control, and a small peak or protrusion appears at the position where the mutant peak may appear, which is a heterozygous sample; 3. When there is a fusion peak, it is a mutant melting point, but a small peak or protrusion appears at the position of the wild peak, which is a heterozygous sample; Heterozygous samples are resistant to rifampicin. It is recommended to repeat the sample test when a heterozygous sample appears to determine the sample resistance.

[0081] Example 3: Verification of the detection capability of the kit

[0082] The detection reagent in Example 1 of the present invention was used to detect Mycobacterium tuberculosis and the national reference plate of rifampicin resistance genes.

[0083] 1. The reagent of the present invention was used to detect the national reference plate of Mycobacterium tuberculosis. The negative reference conformity rate was 100% (15 / 15), the positive reference conformity rate was 100% (15 / 15), the precision reference CV value was <5%, and the detection limit reference S3 (1×10 1 CFU / mL) can be detected. The national reference plate of Mycobacterium tuberculosis is shown in Table 3 below, and the test results are shown in Figures 1 to 3 .

[0084] Table 3 National reference plate for Mycobacterium tuberculosis

[0085]

[0086]

[0087] Second, the reagent of the present invention was used to detect the national reference plate of rifampicin resistance. The compliance rate of the sensitive reference was 100% (10 / 10), the compliance rate of the resistant reference was 100% (17 / 17), and the detection limit of the reference was S (1×10 3 CFU / mL) can be detected. The national reference plate of rifampicin resistance is shown in Table 4 below, and the test results are shown in Figures 4 to 11 ;

[0088] Table 4

[0089]

[0090]

[0091] The method of this embodiment tested 82 sputum samples, of which 56 were positive for Mycobacterium tuberculosis, 26 were negative, and 1 was a discrepancy. The results are as follows:

[0092] Table 5 Result Statistics (I)

[0093]

[0094] The method of this embodiment detected a total of 28 sputum samples positive for Mycobacterium tuberculosis, of which 16 were positive for rifampicin resistance, 12 were negative, and 1 was a differential result. The differential result samples were verified by first-generation sequencing and were uncommon mutation sites for rifampicin resistance, which were not included in the detection sites included in the present invention. The results are statistically as follows:

[0095] Table 6 Result Statistics (II)

[0096]

[0097] The Mycobacterium tuberculosis and rifampicin-resistant reagent, kit and method of the present invention show high sensitivity and specificity when detecting clinical samples and have good clinical application effects.

[0098] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. A nucleic acid composition, characterized in that It comprises: a first primer pair with nucleotide sequences as shown in SEQ ID No.1-2; a second primer pair with nucleotide sequences as shown in SEQ ID No.3-4; and a third primer pair with nucleotide sequences as shown in SEQ ID No.5-6.

2. The nucleic acid composition according to claim 1, characterized in that The nucleic acid composition further comprises: A first probe for detecting the IS6110 gene of Mycobacterium tuberculosis, wherein the nucleic acid sequence of the first probe is shown in SEQ ID No. 7; A second probe for detecting the IS1081 gene of Mycobacterium tuberculosis, wherein the nucleic acid sequence of the second probe is shown in SEQ ID No. 8; A third probe for detecting the first target of rpoB, wherein the nucleic acid sequence of the third probe is shown in SEQ ID No. 9; A fourth probe for detecting the second target of rpoB, wherein the nucleic acid sequence of the third probe is shown in SEQ ID No. 10; A fifth probe for detecting the third target of rpoB, wherein the nucleic acid sequence of the third probe is shown in SEQ ID No. 11; The sixth probe used to detect the fourth target site of rpoB, the nucleic acid sequence of the third probe is shown in SEQ ID No.

12. The 5' ends of the first probe, the second probe, the third probe, the fourth probe, the fifth probe and the sixth probe are independently connected to a fluorescent reporter group, and the 3' ends are independently connected to a fluorescent quencher group.

3. A detection reagent, characterized in that: It comprises the nucleic acid composition according to claim 1 or 2.

4. The detection reagent according to claim 3, characterized in that The detection reagent is used for detecting Mycobacterium tuberculosis complex and rifampicin resistance mutation.

5. A detection kit, characterized in that: It includes: The nucleic acid composition according to claim 1 or 2 or the detection reagent according to claim 3 or 4.

6. The detection kit according to claim, characterized in that The kit also includes: PCR detection reagent.

7. Use of the nucleic acid composition according to claim 1 or 2 in the preparation of a reagent or kit for detecting Mycobacterium tuberculosis complex and rifampicin resistance mutation.

8. A method for detecting Mycobacterium tuberculosis complex and rifampicin resistance mutations, characterized in that: The method comprises: performing PCR detection on a sample using the nucleic acid composition according to claim 1 or 2, the detection reagent according to claim 3 or 4, or the detection kit according to claim 5 or 6.

9. The method for detecting Mycobacterium tuberculosis complex and rifampicin resistance mutation according to claim 8, characterized in that: The PCR instrument used to implement the detection method includes a first fluorescent channel, a second fluorescent channel, a third fluorescent channel and a fourth fluorescent channel, wherein the first fluorescent channel, the second fluorescent channel, the third fluorescent channel and the fourth fluorescent channel are independently a FAM fluorescent channel, a HEX fluorescent channel, a ROX fluorescent channel or a CY5 fluorescent channel; corresponding to the above fluorescent channels, the target target detection signal of the Mycobacterium tuberculosis complex is the amplification curve of the ROX fluorescent channel; the target target detection signals of Mycobacterium tuberculosis combined with rifampicin resistance are the melting curves of the FAM fluorescent channel, the HEX fluorescent channel, the ROX fluorescent channel and the CY5 fluorescent channel respectively; and the internal standard detection signal is the amplification curve of the CY5 fluorescent channel.

10. The method for detecting Mycobacterium tuberculosis complex and rifampicin resistance mutation according to claim 9, characterized in that: Step 1: If the internal standard detects an amplification curve in the CY5 fluorescence channel and CT≤37, it means that this test is valid and proceed to the next step; Step 2: If an S-type amplification curve is detected in the ROX fluorescence channel and Ct≤37, it indicates that the Mycobacterium tuberculosis complex test result is positive; perform the rifampicin resistance analysis step; Step 3: Determine whether the sample has mutated by comparing the difference in melting point of the melting curve between the tested sample and the positive control. When the error between the melting point of the sample in the four channels and the melting point of the positive control does not exceed 1.5°C, it is determined to be wild type, and Mycobacterium tuberculosis in the sample is sensitive to rifampicin; when the absolute value of the difference between the melting point of any channel of the four channels and the melting point of the positive control is 2.0°C or above, it is determined to be mutant type, and Mycobacterium tuberculosis in the sample is resistant to rifampicin.

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