Kit for mycobacterium typing and application thereof

Abstract

The invention relates to a primer probe group, a kit and a method for mycobacterium typing, in particular to the primer probe group, the kit and the method capable of typing 11 mycobacteria by optimizing primer and probe sequences, a Tm value and modification of the Tm value, polymerase and concentration of the polymerase and amplification reaction liquid. The kit or the method disclosed by the invention is high in accuracy, good in specificity, high in sensitivity, low in background signal, low in cost and beneficial to wide application.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a kit for mycobacterium typing and application thereof. Background Art

[0002] Nontuberculous mycobacteria (NTM) are a class of microorganisms that are widely present in the natural environment, some of which can cause diseases in humans and animals. NTM disease is a serious respiratory infection with symptoms similar to tuberculosis, but it is more difficult to treat and is prone to drug resistance. Therefore, accurate typing and detection of NTM is crucial for the prevention and treatment of the disease.

[0003] At present, the typing of NTM mainly relies on traditional biochemical and molecular biological methods, such as ELISA, immunocolloidal gold, DNA sequencing, etc. These methods can accurately identify different types of NTM and provide an important basis for clinical diagnosis and treatment.

[0004] Although the existing NTM typing methods and kits have met clinical needs to a certain extent, there are still some problems and shortcomings. First, traditional biochemical and molecular biological methods are complicated and time-consuming, which is not conducive to rapid diagnosis and treatment. Secondly, although the existing commercial kits can detect a variety of NTMs, their detection range is still limited and the price is high, which is not conducive to their promotion and use in primary medical institutions. Finally, the existing NTM typing methods and kits are often unable to distinguish all types of NTMs. At the same time, most of the existing NTM kits do not contain Mycobacterium tuberculosis and internal references, and cannot completely distinguish Mycobacterium tuberculosis from non-tuberculous mycobacteria, which may lead to misdiagnosis or missed diagnosis.

[0005] Therefore, there is an urgent need in the art to develop a rapid, accurate, simple and economical method for identifying and typing non-tuberculous mycobacteria. Summary of the invention

[0006] The purpose of the present invention is to provide a rapid, accurate, simple and economical kit and method for identifying and typing non-tuberculous mycobacteria.

[0007] In a first aspect of the present invention, a primer probe set for mycobacterium typing is provided, the primer probe set comprising: a first primer probe set, the first primer probe set comprising primers and probes for Mycobacterium marinum, Mycobacterium surga, and Mycobacterium scrofulae,

[0008] The primers and probe for Mycobacterium marinum are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 25, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 26, and a probe having a nucleotide sequence as shown in SEQ ID NO: 27;

[0009] The primers and probe for Mycobacterium surga are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 28, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 29, and a probe having a nucleotide sequence as shown in SEQ ID NO: 30;

[0010] The primers and probe for Mycobacterium scrofulae are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 31, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 32, and a probe having a nucleotide sequence as shown in SEQ ID NO: 33.

[0011] In another preferred embodiment, the primer probe set further includes:

[0012] (a) a second primer-probe set, wherein the second primer-probe set comprises primers and probes for Mycobacterium avium, Mycobacterium chelonae, and Mycobacterium abscessus, respectively;

[0013] (b) a third primer-probe set, comprising primers and probes for Mycobacterium fortuitum and Mycobacterium kansasii, respectively; and

[0014] (c) a fourth primer-probe set, comprising primers and probes for Mycobacterium gordonii, Mycobacterium tuberculosis, and Mycobacterium intracellulare, respectively;

[0015] Wherein, the primers and probe for Mycobacterium avium are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2, and a probe having a nucleotide sequence as shown in SEQ ID NO: 3;

[0016] The primers and probe for Mycobacterium chelonae are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 4, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 6;

[0017] The primers and probe for Mycobacterium abscessus are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8, and a probe having a nucleotide sequence as shown in SEQ ID NO: 9;

[0018] The primers and probe for Mycobacterium fortuitum are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 10, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 11, and a probe having a nucleotide sequence as shown in SEQ ID NO: 12;

[0019] The primers and probe for Mycobacterium kansasii are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 13, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 14, and a probe having a nucleotide sequence as shown in SEQ ID NO: 15;

[0020] The primers and probe for Mycobacterium gordonii are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 16, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 17, and a probe having a nucleotide sequence as shown in SEQ ID NO: 18;

[0021] The primers and probe for Mycobacterium tuberculosis are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 19, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 20, and a probe having a nucleotide sequence as shown in SEQ ID NO: 21;

[0022] The primers and probe for intracellular mycobacterium are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 22, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 23, and a probe having a nucleotide sequence as shown in SEQ ID NO: 24.

[0023] In another preferred embodiment, the primer probe set includes:

[0024] (a) a first primer-probe set, comprising primers and probes for Mycobacterium marinum, Mycobacterium surga, and Mycobacterium scrofulae, respectively;

[0025] (b) a second primer-probe set, comprising primers and probes for Mycobacterium avium, Mycobacterium chelonae, and Mycobacterium abscessus, respectively;

[0026] (c) a third primer-probe set, comprising primers and probes for Mycobacterium fortuitum and Mycobacterium kansasii, respectively; and

[0027] (d) a fourth primer-probe set, comprising primers and probes for Mycobacterium gordonii, Mycobacterium tuberculosis, and Mycobacterium intracellulare, respectively;

[0028] Wherein, the definitions of the first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set are as described above.

[0029] In another preferred embodiment, the probe is modified with a fluorescent group and a quencher group.

[0030] In another preferred embodiment, the primer is modified with locked nucleic acid.

[0031] In another preferred embodiment, the primer comprises a nucleotide modified with a locked nucleic acid.

[0032] In another preferred embodiment, the probe is phosphorylated.

[0033] In another preferred embodiment, the probe comprises a phosphorylated-modified nucleotide.

[0034] In another preferred embodiment, the fluorescent groups of the probes in the same primer probe set are different.

[0035] In another preferred embodiment, the fluorescent group and the quenching group are not modified at the 5' end and the 3' end of the probe.

[0036] In another preferred embodiment, the fluorescent group is selected from the following group: FAM, VIC, ROX, CY5, or a combination thereof.

[0037] In another preferred embodiment, the quenching group is selected from the following group: BHQ, TAMRA, DABCYL, or a combination thereof.

[0038] In another preferred embodiment, the primer-probe set further includes primers and probes for internal references.

[0039] In another preferred embodiment, the primers and probes for the internal reference are primers and probes for the RNaseP fragment of the conserved gene of human.

[0040] In another preferred example, the primers and probe for the internal reference are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 34, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 35, and a probe having a nucleotide sequence as shown in SEQ ID NO: 36.

[0041] In another preferred embodiment, the primer probe set includes:

[0042]

[0043] In a second aspect, the present invention provides a kit, the kit comprising: (i) a first container and a first primer probe set located in the first container;

[0044] (ii) a second container and a second primer probe set located in the second container;

[0045] (iii) a third container and a third primer probe set located in the third container; and

[0046] (iv) a fourth container and a fourth primer probe set located in the fourth container.

[0047] In another preferred example, the kit further comprises a fifth container and a polymerase located in the fifth container, wherein the polymerase comprises Taq enzyme, Vent enzyme and Tth enzyme.

[0048] In another preferred example, the kit further comprises a reaction solution for nucleic acid amplification, and the reaction solution is located in the first container, the second container, the third container and the fourth container or the sixth container.

[0049] In another preferred embodiment, in the reaction solution, the concentration of each upstream primer or downstream primer is independently 20-500 μM, preferably 50-300 μM, more preferably 80-150 μM, for example, 100 μM.

[0050] In another preferred embodiment, in the reaction solution, the concentration of each probe is independently 20-500 μM, preferably 50-300 μM, more preferably 80-150 μM, for example, 100 μM.

[0051] In another preferred embodiment, the kit further comprises reverse transcriptase.

[0052] In another preferred example, the kit further comprises an enhancer, which has a component selected from the group consisting of: MnCl2, SSB single-stranded binding protein, pyrophosphatase, tetramethylammonium chloride, sodium azide, and BSA.

[0053] In another preferred embodiment, the enhancer is located in the seventh container, or added to the reaction solution.

[0054] In another preferred embodiment, when using the kit, the enhancer in the seventh container is added to the reaction solution so that the concentration of each component is 1.0-5.0 g / L MnCl2, 0.1-3 g / L SSB single-stranded binding protein, 0.1-5 g / L pyrophosphatase, 0.1-1 g / L tetramethylammonium chloride, 0.01-1 g / L sodium azide, and 0.1-3 g / L BSA.

[0055] In another preferred embodiment, the concentration of each component of the enhancer in the reaction solution is 1.0-5.0 g / L MnCl2, 0.1-3 g / L SSB single-stranded binding protein, 0.1-5 g / L pyrophosphatase, 0.1-0.8 g / L tetramethylammonium chloride, 0.01-1 g / L sodium azide, and 0.1-3 g / L BSA;

[0056] Preferably, 1.0-3.0 g / L MnCl2, 0.1-1 g / L SSB single-stranded binding protein, 0.1-3 g / L pyrophosphatase, 0.1-0.5 g / L tetramethylammonium chloride, 0.01-0.5 g / L sodium azide, and 0.1-1 g / L BSA.

[0057] In another preferred embodiment, the concentrations of the components of the enhancer in the reaction solution are 2.0 g / L MnCl2, 0.8 g / L SSB single-chain binding protein, 0.5 g / L pyrophosphatase, 0.3 g / L tetramethylammonium chloride, 0.08 g / L sodium azide, and 0.8 g / L BSA.

[0058] In another preferred example, the kit further comprises a lyoprotectant, wherein the lyoprotectant has a component selected from the group consisting of BAS, gelatin, sorbitol, arginine, dextran, and trehalose.

[0059] In another preferred embodiment, the lyophilization protectant is located in the eighth container, or is added to the reaction solution.

[0060] In another preferred embodiment, when using the kit, the lyophilization protectant in the eighth container is added to the reaction solution so that the concentration of each component is 0.1-3 g / L BSA, 0.1%-5% gelatin, 0.1%-5% sorbitol, 0.1-5.0 g / L arginine, 0.1-5.0 g / L dextran, and 0.1-5.0 g / L trehalose.

[0061] In another preferred embodiment, the concentration of each component of the lyoprotectant in the reaction solution is 0.1-3 g / L BSA, 0.1%-5% gelatin, 0.1%-5% sorbitol, 0.1-5.0 g / L arginine, 0.1-5.0 g / L dextran, and 0.1-5.0 g / L trehalose;

[0062] Preferably, 0.1-1 g / L BSA, 0.1%-2% gelatin, 0.1%-2% sorbitol, 0.5-2.0 g / L arginine, 0.5-2.0 g / L dextran, and 0.5-2.0 g / L trehalose.

[0063] In another preferred embodiment, the concentrations of the components of the lyoprotectant in the reaction solution are 0.8 g / L BSA, 1% gelatin, 1% sorbitol, 1.0 g / L arginine, 1.0 g / L dextran, and 1.0 g / L trehalose.

[0064] In another preferred embodiment, the kit further comprises primers and probes for internal reference.

[0065] In another preferred embodiment, the primers and probes for internalization are added to the reaction solution.

[0066] In another preferred embodiment, the kit further comprises a positive control and a negative control.

[0067] In another preferred example, the positive control comprises a mixture of Mycobacterium avium virus-like particles, Mycobacterium chelonae virus-like particles, Mycobacterium abscessus virus-like particles, Mycobacterium fortuitum virus-like particles, Mycobacterium gordonii virus-like particles, Mycobacterium tuberculosis virus-like particles, Mycobacterium intracellulare virus-like particles, Mycobacterium kansasii virus-like particles, Mycobacterium marinum virus-like particles, Mycobacterium surga virus-like particles, Mycobacterium scrofula virus-like particles, and internal reference RNaseP virus-like particles.

[0068] In another preferred embodiment, the negative control substance comprises 0.9% sodium chloride solution.

[0069] The third aspect of the present invention provides a use of the kit according to the second aspect of the present invention for mycobacterium typing.

[0070] In another preferred embodiment, the use is for the purpose of disease diagnosis.

[0071] In another preferred embodiment, the use is for non-disease diagnosis purposes.

[0072] In a fourth aspect, the present invention provides a method for typing mycobacteria, the method comprising the steps of:

[0073] The primer probe set as described in the first aspect of the present invention or the kit as described in the second aspect of the present invention is used to perform a nucleic acid amplification reaction on the sample to be tested, thereby obtaining a test result.

[0074] In another preferred embodiment, the nucleic acid amplification reaction uses a polymerase, and the polymerase includes Taq enzyme, Vent enzyme and Tth enzyme.

[0075] In another preferred embodiment, in the nucleic acid amplification reaction, the enzyme activity ratio of Taq enzyme, Vent enzyme and Tth enzyme is 1-10:1-5:1, preferably 1-8:1-4:1, more preferably 1-5:1-3:1, for example, about 4:2:1.

[0076] In another preferred embodiment, the nucleic acid amplification reaction includes PCR, qPCR, ddPCR or dye-based PCR.

[0077] In another preferred embodiment, the sensitivity of the method is <100 copies / μL, preferably <50 copies / μL, and more preferably <1 copy / μL.

[0078] In another preferred embodiment, the reaction procedure of the nucleic acid amplification reaction is 50°C, 10 minutes, 1 cycle; 95°C, 5 minutes, 1 cycle; 95°C, 10 seconds, 55°C, 40 seconds, 40 cycles.

[0079] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0080] In another preferred embodiment, the judgment criteria of the method are:

[0081] If the amplification curve shows a typical S-shape and the CT value is ≤38, it indicates that the sample to be tested is positive;

[0082] If the CT value is >38 or not detected, it means that the sample to be tested is negative.

[0083] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The results of detecting nontuberculous mycobacteria using the kit of the present invention are shown.

[0085] Figure 2 The detection results at different Tm values ​​are shown.

[0086] Figure 3 The effect of primer and probe modification on the detection results is shown.

[0087] Figure 4 Results of polymerase optimization are shown.

[0088] Figure 5 The effect of the enhancer on the detection results is shown.

[0089] Figure 6 The sensitivity of the method of the present invention is demonstrated.

[0090] Figure 7 This shows the specificity of the method of the present invention. DETAILED DESCRIPTION

[0091] The inventors have conducted extensive and in-depth research, and after a large number of experiments and screening, unexpectedly discovered for the first time a primer probe set, a kit and a method for typing 11 mycobacteria. The inventors optimized the Tm value of the probe, the modification of the primers and probes, and the polymerase and its concentration, and studied the influence of the enhancer on the method of the present invention, and finally obtained the kit of the present invention. On this basis, the present invention was completed.

[0092] the term

[0093] In order to more easily understand the present invention, some technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions, because such methods and conditions can be changed. It should also be understood that the terms used herein are intended only to describe specific embodiments, and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0094] As used herein, the term “comprise” or variations thereof such as “include” or “comprising”, etc., are understood to include the stated elements or components but do not exclude other elements or components.

[0095] The term "about" can refer to a value or composition that is within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0096] As used herein, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include any integer value within the range and fractional values ​​thereof (e.g., one tenth and one hundredth of an integer) where appropriate.

[0097] As used herein, the term "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items.

[0098] Mycobacterium

[0099] Mycobacterium is a kind of elongated slightly curved bacillus, sometimes with branches or filaments, which has been classified as actinomycetes in taxonomy. There are many types of mycobacteria, which can be divided into three categories: Mycobacterium tuberculosis complex, non-tuberculous mycobacteria and Mycobacterium leprae. Among them, the main ones that cause disease to humans are Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium leprae. Mycobacterium involved in the present invention is shown in Table A.

[0100] Table A

[0101] Bacteria Latin name Is it common? Hazards Mycobacterium avium M.avium common high Mycobacterium chelonae M. chelonae Rare high Mycobacterium abscessus M.abscessus common high Mycobacterium fortuitum M.fortuitum Rare high Mycobacterium kansasii M. kansasii common high Mycobacterium gordonii M.gordonae Rare Low Mycobacterium tuberculosis M. tuberculosis common high Mycobacterium intracellulare M.intracellulare common high Mycobacterium marinum M.marinum common high Mycobacterium surga M.szulgai Rare high Mycobacterium scrofula M. scrofulaceum common high

[0102] Table B Grouping and serial numbers of test reagents

[0103]

[0104] Mycobacterium surga

[0105] Mycobacterium surgae is relatively limited in distribution in nature and is considered a rare mycobacterium due to its low clinical isolation rate. Although Mycobacterium surgae is uncommon, its harmfulness should not be underestimated. Currently, it is mainly isolated from specimens of patients with lung or skin infections and is sensitive to most anti-tuberculosis drugs and difficult to treat.

[0106] Primer probe set of the present invention

[0107] The primer probe set of the present invention comprises:

[0108] (a) a first primer-probe set, comprising primers and probes for Mycobacterium marinum, Mycobacterium surga, and Mycobacterium scrofulae, respectively;

[0109] (b) a second primer-probe set, comprising primers and probes for Mycobacterium avium, Mycobacterium chelonae, and Mycobacterium abscessus, respectively;

[0110] (c) a third primer-probe set, comprising primers and probes for Mycobacterium fortuitum and Mycobacterium kansasii, respectively; and

[0111] (d) a fourth primer-probe set, comprising primers and probes for Mycobacterium gordonii, Mycobacterium tuberculosis, and Mycobacterium intracellulare, respectively;

[0112] Wherein, the definitions of the first primer probe set, the second primer probe set, the third primer probe set and the fourth primer probe set are as described in the first aspect of the present invention.

[0113] In a preferred embodiment, the probe is modified with a fluorescent group and a quencher group.

[0114] In a preferred embodiment, the primer is modified with locked nucleic acid.

[0115] In a preferred embodiment, the primer comprises a nucleotide modified with a locked nucleic acid.

[0116] In a preferred embodiment, the primer-probe set further includes primers and probes for internal references.

[0117] In a preferred embodiment, the primer probe set is as shown in Table C.

[0118] Table C

[0119]

[0120]

[0121] The primer probe group of the present invention contains a plurality of probes and primers with different modification modes and different fluorescent labels. The individual bases in the primers are modified by locked nucleic acid, thereby adjusting the primer probe binding free energy distribution so that the binding free energy is sinusoidally distributed, which not only improves the stability of the primer and template binding, but also greatly improves the specificity. The purpose of the multiple modifications of the probe is to reduce the background signal value of the probe itself as much as possible and improve the sensitivity of the detection. In the same reaction system, the primer probe group of the present invention has complementary interference and does not produce cross reaction. In the primer probe group of the present invention, there are corresponding primers and probes for each target. Compared with the method using universal primers, the method of the present invention has higher sensitivity.

[0122] Kit of the present invention

[0123] The kit of the present invention comprises:

[0124] (i) a first container and a first primer probe set located in the first container;

[0125] (ii) a second container and a second primer probe set located in the second container;

[0126] (iii) a third container and a third primer probe set located in the third container; and

[0127] (iv) a fourth container and a fourth primer probe set located in the fourth container.

[0128] In a preferred embodiment, the kit further comprises a reaction solution for nucleic acid amplification, and the reaction solution is located in the first container, the second container, the third container and the fourth container or in the sixth container.

[0129] In a preferred embodiment, the kit also includes a positive control solution and a negative control solution.

[0130] The first container contains primers and probes for identifying Mycobacterium marinum, Mycobacterium surga, and Mycobacterium scrofula, and the probes are respectively labeled with FAM, VIC, and ROX; the second container contains primers and probes for identifying Mycobacterium avium, Mycobacterium chelonae, and Mycobacterium abscessus, and the probes are respectively labeled with FAM, VIC, and ROX; the third container contains primers and probes for identifying Mycobacterium fortuitum and Mycobacterium kansasii, and the probes are respectively labeled with FAM and VIC; the fourth container contains primers and probes for identifying Mycobacterium gordonii, Mycobacterium tuberculosis, and Mycobacterium intracellulare, and the probes are respectively labeled with FAM, VIC, and ROX; each container is provided with an internal reference control, which is a primer and probe for the conserved gene RnaseP fragment of human, and the probe is labeled with CY5.

[0131] In a preferred embodiment, the kit further comprises a fifth container and a polymerase located in the fifth container, wherein the polymerase comprises Taq enzyme, Vent enzyme and Tth enzyme. The amplification efficiency of Vent enzyme is significantly higher than that of Taq enzyme, which is conducive to the rapid accumulation of PCR products in the initial stage of amplification and improves the reaction sensitivity. Tth DNA polymerase has the activity of both reverse transcriptase and DNA polymerase, and has good thermal stability, and reverse transcription can be appropriately introduced during the amplification process, thereby improving the efficiency of reverse transcription and amplification.

[0132] In a preferred embodiment, the positive control solution is a mixture of Mycobacterium avium virus-like particles, Mycobacterium chelonae virus-like particles, Mycobacterium abscessus virus-like particles, Mycobacterium fortuitum virus-like particles, Mycobacterium gordonii virus-like particles, Mycobacterium tuberculosis virus-like particles, Mycobacterium intracellulare virus-like particles, Mycobacterium kansasii virus-like particles, Mycobacterium marinum virus-like particles, Mycobacterium surga virus-like particles, Mycobacterium scrofula virus-like particles, and internal reference RNaseP virus-like particles, and the negative control solution is 0.9% sodium chloride solution.

[0133] The kit of the present invention contains endogenous internal references, which can monitor the entire process of sample collection, nucleic acid extraction, amplification and detection to avoid misjudgment caused by false negative results.

[0134] In a preferred embodiment, the reaction solution further comprises an enhancer selected from the following components: MnCl2 2.0 g / L, SSB single-strand binding protein 0.8 g / L, pyrophosphatase 0.5 g / L, tetramethylammonium chloride 0.3 g / L, sodium azide 0.08 g / L, and BSA 0.8 g / L. MnCl2 and tetramethylammonium chloride help to improve amplification efficiency, SSB single-strand binding protein helps to stabilize the single-strand structure, pyrophosphatase helps to eliminate pyrophosphate generated during the PCR process, thereby improving the continuity of PCR, and BSA helps to improve enzyme thermal stability and enhance the continuity of PCR.

[0135] In a preferred embodiment, the reaction solution further comprises a lyophilization protectant selected from the following components: BSA 0.8 g / L, gelatin 1%, sorbitol 1%, arginine 1.0 g / L, dextran 1.0 g / L, and trehalose 1.0 g / L.

[0136] The main advantages of the present invention include:

[0137] 1. The kit of the present invention can type 11 types of mycobacteria, simplifying the operation process, saving time and having low cost.

[0138] 2. The kit of the present invention contains an endogenous internal reference to avoid misjudgment due to false negative results.

[0139] 3. The primers and probes in the kit of the present invention have different modifications and fluorescent labels, which improve the stability and specificity of the primers binding to the target nucleic acid, reduce the background signal of the probe itself, and improve the sensitivity of detection.

[0140] 4. The kit of the present invention also includes an enhancer. The addition of the enhancer is beneficial to improving the thermal stability of the enzyme, enhancing the sustainability of PCR, and improving the detection signal value and sensitivity.

[0141] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0142] General Methods

[0143] A method for using a nontuberculous mycobacterium typing nucleic acid detection kit comprises the following steps:

[0144] 1. Pretreatment: Take out the positive control and negative control respectively, melt them at room temperature, shake them thoroughly and mix them, then centrifuge them instantly;

[0145] 2. Nucleic acid extraction: nucleic acid extraction is performed simultaneously on the sample to be tested and the positive control and negative control;

[0146] 3. Sample addition: Take out the pre-packed freeze-dried eight-link tube containing the non-tuberculous mycobacterium reaction solution, add 25 μL of the sample to be tested, positive control and negative control nucleic acid, cover the tube tightly, shake evenly and centrifuge instantly;

[0147] 4. Amplification and fluorescence signal detection: placing the PCR reaction tube in a fluorescence quantitative PCR instrument for amplification and fluorescence signal detection;

[0148] 5. Determine the sample test results based on the internal reference test results.

[0149] The non-tuberculous mycobacterium reaction solution in step 3 is pre-packaged and freeze-dried in eight connected tubes, which contain all the components required for fluorescent PCR amplification.

[0150] The amplification and fluorescence signal detection cycle parameters of the fluorescence quantitative PCR instrument in step 3 are as follows: 50°C, 10 minutes, 1 cycle; 95°C, 5 minutes, 1 cycle; 95°C, 10 seconds, 55°C, 40 seconds, 40 cycles.

[0151] Example 1 Design and screening of primers and probes

[0152] 1.1 Primer and probe design for Mycobacterium surga

[0153] Different from the conventional design of primers and probes, multi-copy target regions in the sequence are selected for primer and probe design to ensure the coverage of primer and probe and that primer and probe amplification is not biased in each subtype. Segments with high specificity or conservation are screened out and compared on NCBI to ensure that the comparison results are consistent with the screening results. Multiple different segments are selected for primer and probe design respectively, and the designed primers and probes are screened and tested to finally obtain the primers and probes shown in SEQ ID NO: 28 to 30.

[0154] 1.2 Primer and probe design for other mycobacteria

[0155] According to the types of non-tuberculous mycobacteria published by Genebank, we selected Mycobacterium avium complex, Mycobacterium abscessus and Mycobacterium fortuitum from the main species, and Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium gordonii, Mycobacterium marinum, Mycobacterium intracellulare, Mycobacterium scrofulae, as well as the genome sequences of Mycobacterium tuberculosis and human internal standard genes from the minor species. The differences among the genome sequences were compared and analyzed, and probes and primers were designed.

[0156] The probes for non-tuberculous mycobacteria and Mycobacterium tuberculosis are labeled with FAM / VIC / ROX, respectively, and the probe for the internal reference gene is labeled with CY5, which can identify the typing of Mycobacterium tuberculosis and non-tuberculous mycobacteria in the same reaction system and monitor the entire process of the sample through the internal reference to avoid the occurrence of false negative interpretation results. The probe is an oligonucleotide including a 5' end reporter group and a 3' end quencher group. When the probe is complete, the fluorescence emitted by the reporter group is greatly reduced due to the proximity of the quencher group to the reporter group. During primer extension, the probe bound to the template is cut off by the Taq enzyme (5'→3' exonuclease activity), and the reporter group is separated from the quencher group to generate a fluorescent signal, thereby realizing the detection of Mycobacterium tuberculosis and non-tuberculous mycobacteria typing at the nucleic acid level. Finally, the primers and probes shown in Table C were obtained.

[0157] 1.3 Feasibility Test

[0158] The method used in this example is shown in the general method section, and the primer and probe sequences used are shown in Table C.

[0159] During the PCR reaction, if the reaction well contains an internal reference gene, the labeled CY5 probe should generate a fluorescent signal to monitor sample sampling, sample transportation, nucleic acid extraction, PCR amplification, etc., to avoid false negative test results. Specifically, when the test result is negative, it may be due to unsuccessful sampling, failure of nucleic acid extraction, improper transportation, or errors in the operation process such as liquid preparation or sample addition during PCR amplification. If the reagents at this time include an internal reference and the amplification curve of the internal reference channel is normal, then all of the above possible reasons can be ruled out, thereby avoiding the interpretation of false negative results due to improper operation from sampling to testing.

[0160] The components of each reaction solution are shown in Tables 1a to 1d.

[0161] Table 1a Reaction solution B components

[0162]

[0163]

[0164] Table 1b Reaction solution C components

[0165] Reagent name Add volume (u1) / 50 reactions Mycobacterium fortuitum upstream primer 100 μM 5 Mycobacterium fortuitum downstream primer 100 μM 5 Mycobacterium fortuitum probe (FAM) 100 μM 1 Mycobacterium kansasii upstream primer 100 μM 5 Mycobacterium kansasii downstream primer 100 μM 5 Mycobacterium kansasii probe (VIC) 100 μM 1 Internal reference upstream primer 100 μM 2 Internal reference downstream primer 100 μM 2 Internal reference probe (CY5) 100 μM 0.5 UNG enzyme 5U / uL 1 DNA polymerase 2.5U / uL 40 RNase inhibitor 40U / uL 2.5 Enzyme diluent 154 dATP 100mM 2.5 dCTP 100mM 2.5 dGTP 100mM 2.5 dTTP 100mM 2.5 10xBuffer 125 <![CDATA[MgCl2 25mM]]> 150 Enhancer 100 Lyoprotectant 100 DEPC 280 total 1000

[0166] Table 1c Reaction solution D components

[0167]

[0168]

[0169] Table 1d Reaction solution A components

[0170]

[0171]

[0172] like Figure 1 As shown, the kit of the present invention can identify Mycobacterium avium, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium gordonii, Mycobacterium surga, Mycobacterium marinum, Mycobacterium intracellulare, Mycobacterium scrofula, Mycobacterium tuberculosis and human internal standard genes, which proves the feasibility of the kit and method of the present invention.

[0173] Example 2 Optimization of Primer Probe Tm Value

[0174] In order to improve the signal value of the primer probe, the Tm value of the primer probe was tested in a gradient. Taking the modified combination of the primer probes of Mycobacterium marinum and Mycobacterium chelonae as an example, primer probes with different Tm values ​​were compared and evaluated. The Tm values ​​used in this example are shown in Table 2.

[0175] Table 2

[0176] Tm value 1 Tm value 2 Tm value 3 Mycobacterium chelonae-F 45-47 50-52 54-58 Mycobacterium chelonae-R 45-47 50-52 54-58 Mycobacterium chelonae-P 55-57 60-62 65-70 Mycobacterium marinum-F 45-47 50-52 54-58 Mycobacterium marinum-R 45-47 50-52 54-58 Mycobacterium marinum-P 55-57 60-62 65-70

[0177] The results are as follows Figure 2 As shown in the figure, the amplification effect of Tm value 3 is better than that of Tm value 1 and Tm value 2. After the Tm value is adjusted and optimized, the primer probe signal is improved and the CT value is relatively advanced. Therefore, Tm value 3 is selected for subsequent research.

[0178] Example 3 Modification of primers and probes

[0179] In order to improve the signal value of the primer probe and optimize the amplification curve, three different combinations of primer probe modification methods were set for comparative evaluation, and the modification combinations of primer probes for Mycobacterium gordonii and Mycobacterium intracellulare were used as examples for implementation, and the modifications are shown in Table 3. The method of this embodiment is as described in the general method.

[0180] Table 3

[0181]

[0182]

[0183] The results are as follows Figure 3 As shown, the amplification effect of modification 3 is better than that of modifications 1 and 2. After the locked nucleic acid in the primer is modified, the amplification efficiency of the system is improved. At the same time, the probe is modified, the background signal value is low, and the detection signal value is improved.

[0184] Example 4 Optimization of polymerase

[0185] Four different DNA polymerase combinations were set up, including adding Taq enzyme + Tth enzyme (combination 1, 1.5U + 0.25U), Taq enzyme + Vent enzyme + Tth enzyme (combination 2, 1U + 0.5U + 0.25U), Taq enzyme + Vent enzyme (combination 3, 1.5U + 0.5), and only containing Taq enzyme (combination 4, 2U) to the system for comparative detection. Taking Mycobacterium avium and Mycobacterium chelonae as examples, the experimental steps are as described in the general method.

[0186] like Figure 4 As shown, when Taq enzyme, Vent enzyme and Tth enzyme are added to the system at the same time, the amplification effect of the reaction system is the best.

[0187] Example 5 Effect of Enhancer

[0188] This example studies the effect of adding an enhancer on the amplification results, wherein the enhancer contains 2.0 g / L MnCl2, 0.8 g / L SSB single-stranded binding protein, 0.5 g / L pyrophosphatase, 0.3 g / L tetramethylammonium chloride, 0.08 g / L sodium azide, and 0.8 g / L BSA. Taking Mycobacterium avium and Mycobacterium abscessus as examples, the experimental steps are as described in the general method, wherein the enhancer is added to the reaction solution.

[0189] MnCl2 and tetramethylammonium chloride help improve amplification efficiency, SSB single-stranded binding protein helps stabilize the single-stranded structure, pyrophosphatase helps eliminate pyrophosphate generated during the PCR process, thereby improving the sustainability of PCR, and BSA helps improve enzyme thermal stability and enhance the sustainability of PCR.

[0190] like Figure 5 As shown, regardless of the high concentration sample (10 7 Whether it is a sample with a concentration of 1000 copies / ml or a sample with a low detection limit (1000 copies / ml), the amplification efficiency is significantly improved after adding the enhancer.

[0191] Example 6 Sensitivity and specificity 6.1

[0193] The sensitivity test was performed on samples with a concentration of 1 copy / μl according to the method described in Example 1 and the polymerase and enhancer optimized in Examples 4 and 5.

[0194] The results are as follows Figure 6 As shown, the low concentration detection limit sensitivity of the four-group combination of ABCD (1 copy / μl) and the test detection rate of each channel are 20 / 20. 6.2

[0196] The negative nucleic acid samples extracted from human throat swabs were specifically detected according to the method described in Example 1 and the polymerase and enhancer optimized in Examples 4 and 5.

[0197] like Figure 7 As shown, the specific test detection rate was 0 / 96 (the internal reference was the nucleic acid sample detection rate of 96 / 96).

[0198] The above results show that the method of the present invention has excellent sensitivity and specificity.

[0199] Summarize

[0200] After the optimization of the primers and probes, the optimization of the enzymes and the addition of enhancers, the signal value and sensitivity of the method of the present invention for detecting non-tuberculous mycobacteria are significantly improved.

[0201] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A primer probe set for mycobacterium typing, characterized in that: The primer probe set includes: a first primer probe set, wherein the first primer probe set includes primers and probes for Mycobacterium marinum, Mycobacterium surga, and Mycobacterium scrofulae, The primers and probe for Mycobacterium marinum are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 25, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 26, and a probe having a nucleotide sequence as shown in SEQ ID NO: 27; The primers and probe for Mycobacterium surga are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 28, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 29, and a probe having a nucleotide sequence as shown in SEQ ID NO: 30; The primers and probe for Mycobacterium scrofulae are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 31, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 32, and a probe having a nucleotide sequence as shown in SEQ ID NO:

33.

2. The primer probe set according to claim 1, characterized in that: The primer probe set also includes: (a) a second primer-probe set, wherein the second primer-probe set comprises primers and probes for Mycobacterium avium, Mycobacterium chelonae, and Mycobacterium abscessus, respectively; (b) a third primer-probe set, comprising primers and probes for Mycobacterium fortuitum and Mycobacterium kansasii, respectively; and (c) a fourth primer-probe set, comprising primers and probes for Mycobacterium gordonii, Mycobacterium tuberculosis, and Mycobacterium intracellulare, respectively; Wherein, the primers and probe for Mycobacterium avium are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 1, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 2, and a probe having a nucleotide sequence as shown in SEQ ID NO: 3; The primers and probe for Mycobacterium chelonae are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 4, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 6; The primers and probe for Mycobacterium abscessus are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 7, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 8, and a probe having a nucleotide sequence as shown in SEQ ID NO: 9; The primers and probe for Mycobacterium fortuitum are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 10, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 11, and a probe having a nucleotide sequence as shown in SEQ ID NO: 12; The primers and probe for Mycobacterium kansasii are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 13, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 14, and a probe having a nucleotide sequence as shown in SEQ ID NO: 15; The primers and probe for Mycobacterium gordonii are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 16, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 17, and a probe having a nucleotide sequence as shown in SEQ ID NO: 18; The primers and probe for Mycobacterium tuberculosis are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 19, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 20, and a probe having a nucleotide sequence as shown in SEQ ID NO: 21; The primers and probe for intracellular mycobacterium are: an upstream primer having a nucleotide sequence as shown in SEQ ID NO: 22, a downstream primer having a nucleotide sequence as shown in SEQ ID NO: 23, and a probe having a nucleotide sequence as shown in SEQ ID NO:

24.

3. The primer probe set according to claim 1, characterized in that: The probe is modified with a fluorescent group and a quenching group.

4. The primer probe set according to claim 1, characterized in that: The primers are modified with locked nucleic acid.

5. The primer probe set according to claim 1, characterized in that: The probe is phosphorylated.

6. A kit, characterized in that: The kit comprises: (i) a first container and a first primer probe set located in the first container; (ii) a second container and a second primer probe set located in the second container; (iii) a third container and a third primer probe set located in the third container; and (iv) a fourth container and a fourth primer probe set located in the fourth container.

7. The kit according to claim 6, characterized in that The kit further comprises a fifth container and a polymerase located in the fifth container, wherein the polymerase comprises Taq enzyme, Vent enzyme and Tth enzyme.

8. The kit according to claim 6, characterized in that The kit further comprises an enhancer having a component selected from the group consisting of MnCl2, SSB single-stranded binding protein, pyrophosphatase, tetramethylammonium chloride, sodium azide, and BSA.

9. The use of the kit according to claim 6, characterized in that: Used for mycobacterial typing.

10. A method for mycobacterium typing, characterized in that: The method comprises the steps of: Using the primer probe set as claimed in claim 1 or the kit as claimed in claim 6, a nucleic acid amplification reaction is performed on the sample to be tested to obtain a test result.

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