Mycobacterium typing detection primer group, kit and application

Abstract

The invention discloses a mycobacterium typing detection primer group, a kit and application, the primer group is specifically designed based on 44 pathogenic microorganism genes in mycobacterium infection, can be used for accurate typing of mycobacteria, and is wide in coverage, high in sensitivity and strong in specificity; the method is of great significance to identification of infected strains and diagnosis and treatment of mycobacterium infectious diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of mycobacterium typing detection, in particular to a mycobacterium typing detection primer group, a kit and application. BACKGROUND

[0002] Mycobacterium is a family of 5 genera and more than 200 species of mycobacterium, many of which cause serious diseases in mammals, such as Mycobacterium tuberculosis, which causes a chronic infectious disease. Before the COVID-19 pandemic, tuberculosis was the leading cause of human death from a single infectious disease, and it was also the leading cause of death in HIV carriers and the main cause of death related to antibiotic resistance. Non-tuberculosis mycobacterial infection is also increasing, and NTM infection is very similar to tuberculosis infection in clinical symptoms, which is easily misdiagnosed as tuberculosis in clinical practice, leading to a series of improper treatment methods, which has a certain impact on the patient's condition. Therefore, the identification of the infecting strain is crucial for the diagnosis and treatment of mycobacterial infectious diseases.

[0003] With the rise of next generation sequencing (NGS) technology, it uses high-throughput sequencing technology, which not only reduces costs and speeds up sequencing, but also maintains high accuracy. Targeted NGS (tNGS) first captures specific genes through targeted capture and then performs high-throughput sequencing, which has the characteristic of being able to detect multiple target genes in parallel. However, the second-generation sequencing platform has high construction costs, expensive reagents, long sequencing time, and short read length, making it difficult to cover comprehensive genes, and it requires professional technicians for experimental detection, which greatly limits the sequencing technology in the field of tuberculosis, especially in countries and regions with a heavy burden of tuberculosis prevention and control. The current clinical mycobacterial species identification method based on first-generation sequencing is mostly based on the 16S rRNA region, but the 16S rRNA region has insufficient resolution, and some closely related species cannot be accurately identified. Therefore, developing a highly efficient and accurate mycobacterial identification method and primer group has important application value in the detection of tuberculosis or NTM disease.

[0004] Nanopore sequencing is a new generation of nanopore-based single-molecule real-time electrical signal sequencing technology. It can directly monitor the current changes when nucleic acids pass through protein nanopores in real time, decode these current signals to determine the base sequence, and this sequencing technology can analyze while sequencing, greatly shortening the sequencing time, and the long read length of nanopore sequencing is more suitable for rapid screening of infectious diseases. SUMMARY

[0005] The application provides a mycobacterium typing detection primer group, which realizes high sensitivity, high specificity, accurate and rapid typing detection and is suitable for auxiliary diagnosis of tuberculosis.

[0006] Therefore, the application provides the following solutions. The first aspect of the application is to provide a mycobacterium typing detection primer group, which is designed according to the sequences of 44 pathogenic microorganisms in mycobacterium infection, and the pathogenic microorganisms are as follows:

[0007] The mycobacterium typing detection primer group comprises an amplification primer pair with a nucleotide sequence as shown in SEQ ID NO: 1-270, and the specific corresponding relationship is shown in Table 1.

[0008] Further, the primer group further comprises a reference primer with a nucleotide sequence as shown in SEQ ID NO: 271-272.

[0009] Further, each sequence of the amplification primer pair is sequentially inserted with a stem-loop oligonucleotide and a common sequence from the 5' end to the 3' end direction; the stem-loop oligonucleotide has a length of 4-6 bp and is complementary to the 3' end of the corresponding target sequence.

[0010] Further, the common sequence is shown in SEQ ID NO: 273.

[0011] The second aspect of the application is to provide a mycobacterium typing detection kit, which comprises the primer group according to any one of claims 1-4.

[0012] Preferably, the kit further comprises at least one of a multiplex PCR reaction reagent, a barcode connection PCR reaction reagent and a nanopore library construction reagent.

[0013] Further, the kit further comprises one or more of a nucleic acid extraction reagent, a positive quality control and a negative quality control.

[0014] Further, the nucleic acid extraction reagent is used for nucleic acid extraction or purification of a sample, and the sample is derived from sputum, alveolar lavage fluid or whole blood.

[0015] The third aspect of the application is to provide a preparation method of a mycobacterium sequencing fragment, which comprises the step of performing PCR amplification on a sample nucleic acid by using the primer group according to the first aspect.

[0016] The fourth aspect of the application is to provide a mycobacterium typing detection method, which is not for diagnostic purposes, and the steps comprise: performing amplification on a sample nucleic acid by using the primer group according to the first aspect, then performing sequencing on the amplicon, comparing the sequencing result with a reference gene, and obtaining a detection result.

[0017] Further, the detection method comprises using a primer group to perform first round PCR amplification on the sample nucleic acid, and connecting a barcode for second round PCR amplification; the first round PCR amplification adds a common sequence to the 5' end of each primer in the primer group; And / or, the sequencing is based on a nanopore sequencing method, which can be combined with different platform nanopore sequencing platforms, and is not limited to the ONT sequencing platform of Oxford Nanopore, the Polyseq sequencing platform of Polygenetech, and the Cycloneseq domestic sequencing platform of Huada Smart Manufacturing.

[0018] Compared with the prior art, the present application has the following beneficial effects: The mycobacterium typing detection primer group provided by the present application is specifically designed based on the genes of 44 pathogenic microorganisms in mycobacterium infection, and can be used for accurate typing of mycobacterium, has wide coverage, high sensitivity and strong specificity, and has important significance for identification of infected strains and diagnosis and treatment of mycobacterium infectious diseases. The primer group provided by the present application can be combined with the long read characteristics of nanopore sequencing when used for mycobacterium typing detection, and the identification is more accurate; compared with ordinary PCR and qPCR, the primer group provided by the present application can detect more targets, has higher throughput, lower detection cost, simple operation, short time and convenient detection. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is a gel map of the pathogenic species amplification band in Example 1 of the present application.

[0020] Figure 2 It is a schematic diagram of the hairpin structure in Example 2 of the present application.

[0021] Figure 3 It is a gel map of the hairpin structure amplification band with different base numbers in Example 2 of the present application. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be described below in conjunction with preferred embodiments, obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0023] Example 1 Design and validation of 44 pathogen-specific primers

[0024] 1. Primer pool design: Download all good quality genome sequences of 44 pathogenic microorganisms and humans from the NCBI database, analyze by bioinformatics comparison, select specific sequences within each species and specific to other species, use primer 5.0 to design primers, the Tm value of the designed primers is uniform (60℃±5℃), the length of the amplicon is 200-500bp, the length of the primer is 15-25, the GC content is 40-60%, and the primers with higher scores are preferred. Design 5-10 pairs of primers for each pathogen, sort according to the primer design score, and select the first three pairs of primers of the first species as the initial primer pool, and continuously add primers of other species to the primer pool. If the primers that are easy to generate primer dimers are removed, select other standby primers, and finally the primer pool needs to meet 1-3 pairs of specific primers for each pathogen and 1 pair of human-specific primers.

[0025] 2. Experimental verification: Establish a pathogen reference disc for tuberculosis, and detect all primers in the primer pool by amplification. The primers without amplification bands are removed, and the primers are redesigned and supplemented and verified by experiments.

[0026] Through a large number of repeated experimental tests, the primer pool in Table 1 is finally obtained, Figure 1 which is the corresponding pathogen primer gel map. There are 3 pairs of available primers for each species in the primer pool, and the simultaneous use can improve the sensitivity and specificity of species identification. There are 3 pairs of primers for each species, and the internal reference primers are added, a total of 272 primers in the primer pool. Experiments have proved that there is no cross reaction between them, the primer sequence interference competition is small, and the overall detection sensitivity and specificity are very good.

[0027] Specifically, the primer pool designed for 44 pathogens is shown in Table 1.

[0028] Table 1: Primer pool designed for 44 pathogens

[0029]

[0030]

[0031]

[0032] Example 2 Design of stem-loop oligonucleotide structure

[0033] Design a suitable stem-loop oligonucleotide structure, the 3'-end sequence of the stem-loop primer is complementary to the target, and the schematic diagram of the hairpin structure is shown in Figure 2 . This embodiment selects 4-10bp for experiment, and preferably 4-6bp as a suitable hairpin result, and the results are shown in Table 2.

[0034] Table 2:

[0035] In comparison of the hairpin structure and the glue map without the hairpin structure, it is proved that the hairpin structure is superior to the primer without the hairpin structure in reducing the effect of non-specific amplification, and the results are shown in Figure 3 Table 1, No. 1 is a blank control, No. 2-3 is the original system, No. 4 is a blank control, No. 5-6 is a hairpin 4bp, No. 7 is a blank control, No. 8-9 is a hairpin 6bp, No. 10 is a blank control, No. 11-12 is a hairpin 8bp, No. 13 is a blank control, No. 14-15 is a hairpin 10bp.

[0036] Example 3 Mycobacterium infection pathogen detection primer combination

[0037] A round of reaction uses a combination of multiple specific reaction sequences, each sequence from 5' end to 3' end consists of a segment sequence, a segment sequence, and a segment sequence, wherein the segment sequence is a stem-loop oligonucleotide structure, the 3'-end sequence of the primer is complementary to the 6 bases of the target, the segment sequence is a common sequence (SEQ ID NO: 325), and the segment sequence is the forward and reverse sequence of 135 pairs of specific primers (SEQ ID NO: 1~270), a total of 270 sequences are combined to obtain 270 sequences, which are mixed according to a certain concentration and then used. The final concentration of each primer system is 5nM~100nM, preferably 10nM concentration, and the 3 pairs of primers of Mycobacterium tuberculosis complex are given as an example, as shown in Table 3.

[0038] Table 3:

[0039] In the primers described in Table 3, the underlined wave line is the stem-loop oligonucleotide structure sequence, the underlined straight line is the common sequence, and the unlined part is the specific primer sequence.

[0040] Example 4 Kit composition

[0041] The kit for detecting mycobacterium typing includes amplification reaction solution, primer pool, and label reagent, and the kit components are shown in Table 4.

[0042] Table 4:

[0043] Example 5 Method flow for detecting mycobacterium infection pathogen

[0044] (1) Sample pretreatment and nucleic acid extraction

[0045] Take 200 μl of the sputum sample to be tested, and inactivate the clinical sample before extraction. Place it at 60°C for 30 min. Use nucleic acid extraction or purification reagents (Benay Medicine TQ006D-50, TQ007D-64). Refer to the corresponding instructions for the specific extraction method.

[0046] (2) One round of multiple target-specific reactions

[0047] Extract the nucleic acid and positive and negative controls, and perform multiple PCR amplification according to the following reaction system: One round of multiple amplification system A of the kit:

[0048] The reaction conditions of amplification system A are as follows: annealing and extension temperature 53-60°C, preferably 60°C; annealing and extension time 30 s-5 min, preferably 3 min.

[0049] (2) Magnetic bead purification of PCR products

[0050] 2.1 Vortex the magnetic beads, and stand at room temperature for 30 min.

[0051] 2.2 Take a new 200 μl eight-tube tube, and purify 20 μl of PCR product system A from the previous step. Add 20 ul of water, and then add 32 μl (0.8x) of magnetic bead purification.

[0052] 2.3 Vortex, stand at room temperature for 5 min, and centrifuge. Place the sample tube in the magnetic stand for 2 min, and carefully remove the supernatant after the solution is completely clarified.

[0053] 2.4 Keep the sample tube in the magnetic stand at all times, add 200 μl of freshly prepared 80% ethanol to rinse the magnetic beads, and stand at room temperature for 30-60 s. Carefully remove the supernatant.

[0054] 2.5 Repeat 2.4 once, for a total of two rinses.

[0055] 2.6 Keep the sample in the magnetic stand at all times, and dry the magnetic beads at room temperature for about 2-3 min.

[0056] 2.7 Take the sample out of the magnetic stand, add 10 μl of elution buffer, mix well, stand at room temperature for 2-3 min, centrifuge briefly, place in the magnetic stand for 2 min, and carefully pipette 9 μl of the supernatant into a new centrifuge tube after the solution is clarified. If not used immediately, store at -20°C.

[0057] (3) Second round of tag ligation PCR reaction

[0058] Take a round of reaction product according to the following table for label connection PCR reaction, different samples use different labels.

[0059] The second round of multiplex amplification system B of the kit:

[0060] The reaction conditions of amplification system B: through experimental test, the annealing temperature is 55-65℃, preferably 60℃; the annealing time is 30s-2min, preferably 30s; the extension time is 30s-1min, preferably 40s:

[0061] (4) Magnetic bead purification of PCR product

[0062] 4.1 Magnetic bead vortex mixing, room temperature standing for 30 min.

[0063] 4.2 Take a new 200 μl eight connected tube, purify 15 μl PCR product system B of the above step alone, add 25 ul water, then add 32 μl (0.8x) magnetic bead purification.

[0064] 4.3 Vortex mixing, room temperature standing for 5 min, instant centrifugation, place the sample tube on the magnetic stand for 2 min, after the solution is completely clarified, carefully remove the supernatant.

[0065] 4.4 Keep the sample tube in the magnetic stand all the time, add 200 μl of newly prepared 80% ethanol to rinse the magnetic beads, room temperature for 30-60 s, carefully remove the supernatant.

[0066] 4.5 Repeat 2.4 once, a total of two times.

[0067] 4.6 Keep the sample in the magnetic stand all the time, dry the magnetic beads at room temperature for about 2-3 min.

[0068] 4.7 Take the sample out of the magnetic stand, add 10 μl of elution buffer, mix well, room temperature standing for 2-3 min, short centrifugation, place on the magnetic stand for 2 min, after the solution is clarified, carefully pipette 9 μl of supernatant into a new centrifuge tube. If not used immediately, store at -20℃.

[0069] 4.8 Take 1 μl of purified product to detect the concentration with Qubit dsDNA HS Assay Kit.

[0070] (5) Library construction and sequencing

[0071] After the product is pooled, the library is constructed according to the nanopore sequencing process. The detection kit can be used for sequencing with different nanopore platforms, including but not limited to the ONT sequencing platform of Oxford Nanopore, Pintbio, Huada Smartech, and all domestic nanopore sequencing platforms. The machine operation instruction of the nanopore sequencer is followed to perform the machine operation.

[0072] (6) Bioinformatics analysis

[0073] The data after the machine is analyzed by bioinformatics, compared with the pathogen database, and the detection results are analyzed.

[0074] Example 6

[0075] A 44-pathogen reference disc is established, and the reference disc is detected by the above method. The detection results are shown in Table 5.

[0076] Table 5: Detection results of 44-pathogen reference disc

[0077]

[0078]

[0079] The primer numbers "P1, P2, P3..." are consistent with the arrangement order of each pathogen primer pair in Table 1. For example, the Mycobacterium tuberculosis complex primer P1 represents the primer pair with sequence 1-2, and the sequence is classified accordingly.

[0080] Example 7

[0081] The clinical sample compliance verification is performed in this example, and the test results are shown in Table 6. The results show that the detection method is consistent with the sample culture results, and the positive and negative coincidence rate is 100%.

[0082] Table 6: Consistency of detection of some positive species

[0083] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A primer set for mycobacterium typing detection, characterized in that: The amplification primer pair includes nucleotide sequences as shown in SEQ ID NO: 1-270.

2. The primer set according to claim 1, characterized in that Internal reference primers are also included, and the nucleotide sequences are shown in SEQ ID NOs: 271-272.

3. The primer set according to claim 1, wherein Each sequence of each amplification primer pair is sequentially inserted with a stem-loop oligonucleotide and a common sequence from the 5' end to the 3' end; the stem-loop oligonucleotide is 4-6 bp in length and is complementary to the 3' end of the target sequence corresponding to the primer.

4. The primer set according to claim 3, characterized in that The common sequence is shown as SEQ ID NO:

273.

5. A mycobacterium typing detection kit, characterized in that: The invention comprises the primer set according to any one of claims 1 to 4.

6. The detection kit according to claim 5, characterized in that The kit further comprises at least one of a multiplex PCR reaction reagent, a barcode ligation PCR reaction reagent, and a nanopore library construction reagent.

7. The detection kit according to claim 5, characterized in that The kit further comprises one or more of a nucleic acid extraction reagent, a positive quality control and a negative quality control.

8. A method for preparing a Mycobacterium sequencing fragment, characterized in that: The method comprises the step of performing PCR amplification on the sample nucleic acid using the primer set according to claim 1.

9. A method for mycobacterial typing detection for non-diagnostic purposes, characterized in that the steps include: The sample nucleic acid is amplified using the primer set described in claim 1, and then the amplicon is sequenced. The sequencing result is compared with the reference gene to obtain the test result.

10. The method according to claim 9, characterized in that The detection method comprises performing a first round of PCR amplification on the sample nucleic acid using a primer set, and connecting a barcode to perform a second round of PCR amplification; the first round of PCR amplification adds a common sequence to the 5' end of each primer in the primer set; And / or, the sequencing is based on nanopore sequencing.

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