CN113005212A

CN113005212A - Primer probe composition, kit and method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria

Info

Publication number: CN113005212A
Application number: CN202110469325.4A
Authority: CN
Inventors: 朱海涛; 夏江
Original assignee: Pilot Gene Technologies Hangzhou Co ltd
Current assignee: Pilot Gene Technologies Hangzhou Co ltd
Priority date: 2021-04-28
Filing date: 2021-04-28
Publication date: 2021-06-22

Abstract

The invention belongs to the field of biology, and particularly relates to a primer probe composition, a kit and a method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria. The invention discloses a primer probe composition, which comprises a nucleotide sequence shown as SEQ ID NO: 1-6. Also disclosed are kits comprising the primer probe compositions, and methods of using the primer probe compositions or kits for the detection of mycobacterium tuberculosis and nontuberculous mycobacteria. The kit disclosed by the invention is based on a multiple liquid drop digital PCR platform, and the platform has the characteristics of high sensitivity and multiple applicability; then, screening reasonable primer pair combinations through the combination optimization of the autonomous primer pairs; can quickly and sensitively detect the mycobacterium tuberculosis and nontuberculous mycobacteria.

Description

Primer probe composition, kit and method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria

Technical Field

The invention belongs to the field of biology, and particularly relates to a primer probe composition, a kit and a method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria.

Background

Mycobacterium tuberculosis (m. tuberculosis), commonly known as tubercle bacillus, is the causative agent of tuberculosis. It can invade all organs of the body, but pulmonary tuberculosis is the most common. Tuberculosis remains an important infectious disease to date. It is reported by WHO that about 800 new cases occur each year, and at least 300 million people die from the disease. The mortality rate of 300 people per 10 million before China is established, the death rate of people living in various diseases is the first, the living level of people is improved after China is established, the sanitary condition is improved, group prevention and mass control are particularly developed, children are generally inoculated with BCG, and the morbidity and mortality of tuberculosis are greatly reduced. However, it should be noted that some parts of the world have an increasing incidence due to aids, drug abuse, application of immunosuppressive agents, alcohol abuse, poverty and the like.

Nontuberculous bacteria, i.e., nontuberculous mycobacteria, are widely present in nature in soil, dust, water, fish and poultry, and are mainly transmitted by acquiring infections such as sewage from the environment, and human-to-human infection is rare. Such mycobacteria are generally less pathogenic to humans than mycobacterium tuberculosis, but can lead to lesions if susceptible factors are present that cause a dysfunction of the host's local or systemic immune function.

The existing detection methods for mycobacterium tuberculosis and nontuberculous mycobacteria include bacterial culture method, high performance liquid chromatography, DNA sequencing and membrane strip hybridization technology, qPCR and the like.

The prior art has the main defects of relatively complex and time-consuming operation, insufficient sensitivity, difficulty in accurately distinguishing the mycobacterium tuberculosis from the nontuberculous mycobacterium and the like.

Disclosure of Invention

The invention aims to find a novel kit which can be used for quickly and sensitively detecting mycobacterium tuberculosis and nontuberculous mycobacterium.

The digital PCR technology is a third generation PCR technology developed on the basis of the qPCR technology, can theoretically realize amplification detection on a single copy of a target nucleic acid fragment, and is the current nucleic acid detection technology with the highest sensitivity.

The kit provided by the invention designs multiple primer pair combinations, can simultaneously qualitatively detect mycobacterium tuberculosis, nontuberculous mycobacterium and reference genes, supports high-throughput, rapid, accurate and ultrahigh-sensitivity detection, and has extremely high clinical application value.

Specifically, the technical scheme of the invention is as follows:

in a first aspect, the present invention discloses a primer probe composition, comprising:

nucleotide sequences of a primer group and a probe for detecting mycobacterium tuberculosis are respectively shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 is shown in the specification;

the nucleotide sequence of the primer group for detecting nontuberculous mycobacteria is shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: and 6.

It is understood that nucleotide sequence variants having more than 90% similarity and functional identity to the above-described nucleotide sequences are within the scope of the present invention.

In a second aspect, the invention discloses a product comprising the primer probe composition.

Preferably, the product is a kit; more preferably, the kit is a kit for detecting mycobacterium tuberculosis and nontuberculous mycobacteria.

More preferably, the kit comprises a detection reagent.

The third aspect of the invention discloses a method for detecting mycobacterium tuberculosis and nontuberculous mycobacteria, which uses the primer probe composition or the product for detection.

Preferably, the method further comprises an internal control system, wherein the internal control system comprises a nucleotide sequence shown as SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 10, or a fragment thereof.

Preferably, the detection is based on a multiplex digital PCR platform.

The term "multiplex digital PCR" refers to a digital PCR reaction in which two or more pairs of primers are added to the same digital PCR reaction system to simultaneously amplify multiple nucleic acid fragments. The multiplex PCR formed by organically combining the multiplex PCR technology and the digital PCR technology can greatly improve the multiplex of the digital PCR according to different fluorescence degrees of the DNA probe, different numbers of cycles of DNA amplification and simultaneous use of multiple marked fluorescence, and the multiplex can reach more than 20-50, namely more than 20-50 digital PCR reactions can be simultaneously carried out in one PCR reaction unit.

More preferably, the method comprises the following steps:

(1) obtaining a sample nucleic acid;

(2) preparing a digital PCR reaction solution;

(3) preparing a liquid drop chip;

(4) and (4) after the liquid drop chip amplification program is operated, analyzing by adopting a biochip reader and outputting a report.

Preferably, the amplification procedure comprises, in order: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 60 seconds, and circulation for 40 times.

It should be understood that the amplification procedure is not limited to the above-mentioned amplification procedure, and those skilled in the art can select an appropriate amplification procedure according to the need and fall within the scope of the present invention.

It is emphasized that it is within the ability of the person skilled in the art to select a suitable method according to the teachings of the present invention, and not limited to the solution described above.

The fourth aspect of the invention discloses the application of the primer probe composition, the product or the method in the clinical field.

Compared with the prior art, the invention has the following remarkable advantages and effects:

the method and the kit for detecting the mycobacterium tuberculosis and the nontuberculous mycobacterium, provided by the invention, have the following advantages: 1. the probe is reasonable in design, multiple PCR can be carried out by utilizing a multiple digital PCR platform, and multiple targets can be detected by one fluorescence channel in one reaction system; 2. the components of the kit in the invention are optimized, so that the mycobacterium tuberculosis gene and the non-mycobacterium tuberculosis gene can be detected quickly and sensitively; 3. the multiplex digital PCR platform can realize the qualitative detection function on the ultramicro nucleic acid sample, and the detection sensitivity can be as low as less than 10 copies.

Detailed Description

The technical solutions of the present invention are described in detail below with reference to examples, but the present invention is not limited to the scope of the examples.

The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions. The reagents and starting materials used in the present invention are commercially available.

Example 1

This example discloses a method for simultaneous detection of Mycobacterium tuberculosis and nontuberculous mycobacteria.

Firstly, experimental materials:

1. sample requirements:

1.1 Mycobacterium tuberculosis DNA: positive DNA detected by Xpert;

1.2 non-tuberculous mycobacterial DNA: using a generation of sequencing verified mycobacterium avium DNA;

1.3 Normal human DNA: genomic DNA extracted from buccal swabs;

1.4 bacterial DNA: DNA extracted from an inactivation positive strain purchased from the company.

2. The experimental flow chart is as follows:

extracting free nucleic acid from a sample → configuring digital PCR reaction solution → generating liquid drop chip → amplifying → reading → analyzing the result and outputting a report

II, an experiment step:

samples were tested using a digital PCR platform.

The digital PCR detection process (configuring digital PCR reaction solution, droplet chip generation and amplification process) is as follows:

designing a group of primer probes according to the sequence of the mycobacterium tuberculosis IS6110 gene; designing two groups of non-tuberculous mycobacterium detection systems (five non-tuberculous mycobacterium shared primer probes) according to the 16S sequences of the tuberculous mycobacterium and the non-tuberculous mycobacterium (mycobacterium avium, mycobacterium intracellulare, mycobacterium abscessus, mycobacterium cheloniae and mycobacterium kansasii); the artificially synthesized gene segment and primer probe are used as internal control system. The specific information is shown in table 3 below:

TABLE 3

2. Prepare 15 mul drop PCR detection system, the final concentration of the specific system formula is shown in Table 4:

TABLE 4 formulation

3. Adding the extracted nucleic acid template into different systems, uniformly mixing, and preparing a positive control and a negative control of an experiment at the same time;

4. adding the prepared reaction system into a sample adding hole of a droplet chip according to an SOP process; the chip was placed in the sample preparation instrument and the instrument was started to generate droplets.

5. Putting the chip into a chip amplification instrument; setting a liquid drop chip amplification program and operating;

and (3) amplification procedure: 5min at 98 ℃ [ 98 ℃ 15s, 60 ℃ 1min ] 40.

Fourthly, reading by a droplet chip, analyzing results and reporting the flow:

1. after the amplification is finished, taking out the chip rack and placing the chip rack on a chip placing table of a digital PCR reader, opening GenePMS software, selecting a corresponding fluorescence detection channel, starting chip scanning and analyzing the result;

2. and (6) data analysis and report output.

Fifth, the detection result

1. The results of the M.tuberculosis/M.nontuberculosa specificity test are shown in Table 5 below:

TABLE 5

2. The results of the tests for specificity to common pathogenic bacteria are shown in table 6 below:

TABLE 6

3. The human genome specificity test is shown in table 7 below:

TABLE 7

4. The results show that

The minimum detection limit of Mycobacterium tuberculosis is 0.2 copies/mu L, and the minimum detection limit of nontuberculous mycobacteria is 0.4 copies/mu L.

5. Conclusion

The detection kit disclosed by the invention has normal specificity, and the mycobacterium tuberculosis and nontuberculous mycobacterium system have no cross reaction; the detection system of the mycobacterium tuberculosis and the nontuberculosis has no cross reaction with 16 common pathogenic bacteria; the detection system of the mycobacterium tuberculosis and the nontuberculous mycobacterium has no cross reaction with the human genome DNA.

The multiple digital PCR system can qualitatively detect the mycobacterium tuberculosis, the nontuberculous mycobacterium and the reference gene simultaneously, and has higher sensitivity.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

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Claims

1. A primer probe composition, comprising:

2. A product comprising the primer probe composition of claim 1.

3. The product of claim 2, wherein the product is a kit; preferably, the kit is a kit for simultaneously detecting mycobacterium tuberculosis and nontuberculous mycobacteria.

4. The product of claim 3, wherein the kit comprises a detection reagent.

5. A method for simultaneous detection of Mycobacterium tuberculosis and nontuberculous mycobacteria, which is characterized by using the primer probe composition of claim 1 or the product of claims 2 to 4 for detection.

6. The method of claim 5, further comprising an internal control system, wherein the internal control system comprises a nucleotide sequence set forth in SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 and SEQ ID NO: 10, or a fragment thereof.

7. The method of claim 5, wherein the detection is based on a multiplex digital PCR platform.

8. The method of claim 7, comprising the steps of:

(1) obtaining a sample nucleic acid;

(2) preparing a digital PCR reaction solution;

(3) preparing a liquid drop chip;

9. The method of claim 7, wherein the amplification procedure comprises, in order: pre-denaturation at 98 ℃ for 5 min; denaturation at 98 ℃ for 15 seconds, annealing at 60 ℃ for 60 seconds, and circulation for 40 times.

10. Use of the primer probe composition according to claim 1, the product according to claims 2-4 or the method according to claims 5-9 in clinical fields.