CN112538539B - Kit and system for detecting mycobacterium avium - Google Patents
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Abstract
The present disclosure relates to a kit and a system for detecting mycobacterium avium, the kit comprising a reagent for detecting a molecular marker, wherein the molecular marker comprises at least one of nikA gene, ddpC gene, yejF gene, rrl G2368T locus, rrl T3066A locus, and acpM C177T locus. By identifying the above molecular markers, the present disclosure enables accurate detection of mycobacterium avium.
Description
Technical Field
The present disclosure relates to the field of biotechnology, in particular to a kit for mycobacterium avium detection, use of a reagent for detecting a molecular marker in the preparation of a kit for mycobacterium avium detection, and a system for mycobacterium avium detection.
Background
Nontuberculous mycobacteria (NTM) refer to mycobacteria other than Mycobacterium tuberculosis complex (Mtb) and Mycobacterium leprae, and are widely present in natural environments. About one third of the NTM can cause infection in animals or humans, for example, mycobacterium kansasii, mycobacterium avium, mycobacterium intracellulare, mycobacterium abscessus, mycobacterium cheloniae, and the like.
NTM infection is common in patients with structural lung diseases, and after the infection of human bodies, NTM often invades lung tissues of the patients, so that bronchial tissues and alveolar tissues of the patients are progressively damaged, symptoms similar to pulmonary tuberculosis are caused, the respiratory function of the patients is influenced, and the death of the hosts can be caused in severe cases. In recent years, the infection rate of NTM is rising year by year, and a plurality of researches show that NTM can be infected from person to person, so that NTM detection is carried out timely and accurately, and the method has important social and clinical values.
In the related art, NTM detection mostly relies on target gene sequencing and mass spectrometry detection, wherein common target genes include genes encoding 16S rRNA, 16S-23S rRNA intergenic region (ITS), RNA polymerase β subunit (rpoB), heat shock protein 65(hsp65), and DNA helicase (gyrA/gyrB), sodA gene, recA gene, and the like.
However, in the process of implementing the present disclosure, the inventors found that the resolution of the existing NTM detection technology is low, and NTM species with close relativity cannot be accurately resolved.
Disclosure of Invention
The purpose of the present disclosure is to solve the problem of low resolution in the existing NTM detection technology, and provide a kit and a system capable of accurately detecting mycobacterium avium.
In order to accomplish the above objects, the present disclosure provides, in a first aspect, a kit for detection of mycobacterium avium, the kit comprising a reagent for detecting a molecular marker, wherein the molecular marker comprises at least one of nikA gene, ddpC gene, yejF gene, rrl G2368T site, rrl T306 3066A site, and acpM C177T site.
Optionally, the reagent for detecting a molecular marker comprises a primer pair capable of specifically amplifying the molecular marker, and/or a probe capable of specifically hybridizing to the molecular marker.
Optionally, the primer pair comprises at least one pair of primers shown by SEQ ID NO 1-2, SEQ ID NO 3-4, SEQ ID NO 5-6, SEQ ID NO 7-8, SEQ ID NO 9-10 and SEQ ID NO 11-12.
In a second aspect, the present disclosure provides use of a reagent for detecting a molecular marker including at least one of nikA gene, ddpC gene, yejF gene, rrl G2368T site, rrl T306 3066A site, and acpM C177T site in the preparation of a kit for detection of mycobacterium avium.
Optionally, the reagent for detecting a molecular marker comprises a primer pair capable of specifically amplifying the molecular marker, and/or a probe capable of specifically hybridizing to the molecular marker.
Optionally, the primer pair comprises at least one pair of primers shown by SEQ ID NO 1-2, SEQ ID NO 3-4, SEQ ID NO 5-6, SEQ ID NO 7-8, SEQ ID NO 9-10 and SEQ ID NO 11-12.
In a third aspect, the present disclosure provides a system for mycobacterium avium detection, the system comprising a sequencing device, a computing device, and an output device;
the sequencing device is used for carrying out nucleic acid sequence sequencing on the total nucleic acid of the sample to be tested to obtain a total nucleic acid sequence;
the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the determination of:
if the total nucleic acid sequence contains at least one of nikA gene sequence, ddpC gene sequence, yejF gene sequence, rrl G2368T site sequence, rrl T3066A site sequence and acpM C177T site sequence, judging that the mycobacterium avium exists in the sample to be detected;
the output device is used for outputting the judgment result of the computing device.
Optionally, the system further includes a nucleic acid extraction device, and the nucleic acid extraction device is configured to perform nucleic acid extraction on the sample to be detected, so as to obtain total nucleic acid of the sample to be detected.
Optionally, the nucleic acid extraction device comprises a nucleic acid extractor and/or a nucleic acid extraction kit.
Optionally, the sequencing device comprises at least one of a Sanger sequencing platform, Illumina Novaseq, HiSeq Xten, HiSeq2500/2000/4000 sequencing platform.
Through the technical scheme, the accurate detection of the mycobacterium avium can be at least partially realized by identifying molecular markers such as nikA gene, ddpC gene, yejF gene, rrl G2368T site, rrl T3066A site, acpM C177T site and the like.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
A first aspect of the present disclosure provides a kit for mycobacterium avium detection, the kit comprising a reagent for detecting a molecular marker, wherein the molecular marker comprises at least one of nikA gene, ddpC gene, yejF gene, rrl G2368T site, rrl T3066A site, and acpM C177T site.
The inventor of the present disclosure finds that specific molecular markers which are different from other non-tuberculous mycobacteria or tuberculous mycobacteria exist in the mycobacterium avium, namely nikA gene, ddpC gene, yejF gene, rrl G2368T site, rrl T3066A site and acpM C177T site, and by detecting the molecular markers, the mycobacterium avium can be accurately distinguished from other non-tuberculous mycobacteria or tuberculous mycobacteria, so that accurate detection of the mycobacterium avium is at least partially realized.
Specifically, the gene numbers of the above-mentioned molecular markers are shown in Table 1.
TABLE 1
| Molecular marker | Gene numbering |
| nikA gene | 2718029 |
| ddpC gene | 2720266 |
| yejF gene | 2720026 |
| rrl G2368T site | 2700466 |
| rrl T3066A site | 2700466 |
| acpM C177T site | 15609381 |
According to the present disclosure, the reagent for detecting a molecular marker may be selected within a certain range, for example, the reagent for detecting a molecular marker may include a primer pair capable of specifically amplifying the molecular marker, and/or a probe capable of specifically hybridizing to the molecular marker.
According to the present disclosure, the primer pair and the probe may be selected within a certain range, and both the primer pair and the probe satisfying the above requirements may be used in the present disclosure. As a preferred embodiment of the present disclosure, the primer pair may include at least one pair of primers shown by SEQ ID NO. 1-2, SEQ ID NO. 3-4, SEQ ID NO. 5-6, SEQ ID NO. 7-8, SEQ ID NO. 9-10, and SEQ ID NO. 11-12.
Wherein the target molecule markers and nucleotide sequences of the primer pairs are shown in Table 2.
TABLE 2
A second aspect of the present disclosure provides use of a reagent for detecting a molecular marker including at least one of nikA gene, ddpC gene, yejF gene, rrl G2368T site, rrl T3066A site, and acpM C177T site in preparing a kit for detection of mycobacterium avium.
Alternatively, the reagent for detecting a molecular marker may include a primer pair capable of specifically amplifying the molecular marker, and/or a probe capable of specifically hybridizing to the molecular marker.
Optionally, the primer pair can comprise at least one pair of primers shown in SEQ ID NO 1-2, SEQ ID NO 3-4, SEQ ID NO 5-6, SEQ ID NO 7-8, SEQ ID NO 9-10 and SEQ ID NO 11-12.
A third aspect of the present disclosure provides a system for mycobacterium avium detection, the system comprising a sequencing device, a computing device, and an output device; the sequencing device is used for carrying out nucleic acid sequence sequencing on the total nucleic acid of the sample to be tested to obtain a total nucleic acid sequence; the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the determination of: if the total nucleic acid sequence contains at least one of nikA gene sequence, ddpC gene sequence, yejF gene sequence, rrl G2368T site sequence, rrl T3066A site sequence and acpM C177T site sequence, judging that the mycobacterium avium exists in the sample to be detected; the output device is used for outputting the judgment result of the computing device.
Optionally, the system may further include a nucleic acid extraction device, where the nucleic acid extraction device is configured to extract nucleic acid from the sample to be tested, so as to obtain total nucleic acid of the sample to be tested.
Optionally, the nucleic acid extraction device may comprise a nucleic acid extractor and/or a nucleic acid extraction kit.
Alternatively, the sequencing device may be, for example, a second generation sequencing device, for example, the sequencing device may comprise at least one of the Sanger sequencing platform, Illumina Novaseq, HiSeq Xten, HiSeq2500/2000/4000 sequencing platform.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
The raw materials, reagents, instruments and equipment referred to in the examples of the present disclosure may be purchased, unless otherwise specified.
Example 1
This example serves to illustrate the determination of biomarkers provided by the present disclosure for detection of mycobacterium avium.
81 NTM strains having a complete genome were collected as discovery groups including 7 M.kansasii, 27 M.avium, 6 M.intracellulare, 5 M.tortoise and 36 M.abscessus.
In addition, 384 NTM strains with the original sequencing reads were collected as the first validation set, which included 14 mycobacterium kansasii, 111 mycobacterium avium, 26 mycobacterium intracellulare, 33 mycobacterium cheloni, and 200 mycobacterium abscessus.
And performing comparative genomics analysis on the genome data of the discovery group, and determining the distinguishing genes and the distinguishing SNPs sites in the genome of the 27 strains of mycobacterium avium, which are distinguished from the genomes of other NTM strains, namely nikA gene, ddpC gene, yejF gene, moxC gene, hldE gene, ino 1G 832A site, clpB G2124C site, rrl G447A site, rrl T455A site, rrl G2368T site, rrl T3066A site, Rv 1461G 2133A site, acpM C177T site, Rv 2402G 570C site, rpsK C90G site and pks 13G 672C site.
Aiming at the distinguishing genes and the distinguishing SNPs sites, X2 test and random forest prediction model analysis are carried out in a first verification group, specific genes and specific SNPs sites which are different from the genomes of other NTM strains in the genomes of the mycobacterium avium are screened out from the distinguishing genes and the distinguishing SNPs sites, namely nikA genes, ddpC genes, yejF genes, rrl G2368T sites, rrl T3066A sites and acpM C177T sites, and the specific genes and the specific SNPs sites are used as biomarkers for detecting the mycobacterium avium.
Example 2
This example is used to verify the effect of the biomarkers provided by the present disclosure on the detection of mycobacterium avium.
164 NTM strains with original sequencing reads were collected as a second validation set, including 6 M.kansasii, 48 M.avium, 11 M.intracellulare, 14 M.tortoise and 85 M.abscessus.
And (3) respectively carrying out total nucleic acid extraction on the five mycobacteria by using a nucleic acid extraction kit to obtain the total nucleic acid of each mycobacterium. Then, the total nucleic acid of each mycobacterium obtained above is subjected to second-generation sequencing to obtain the total nucleic acid sequence of each mycobacterium.
Based on at least a part of the molecular markers determined in example 1, the presence or absence of the corresponding molecular marker sequence in the total nucleic acid sequence of each of the above-mentioned mycobacteria is determined, and the mycobacteria containing the corresponding molecular marker sequence is determined to be mycobacterium avium, and the detection result is obtained. The molecular markers targeted for each detection are shown in table 3.
TABLE 3
And (3) respectively carrying out ROC analysis on the detection results of the detections 1-9, and determining the specificity, sensitivity and AUC value of each detection, wherein the results are shown in Table 4.
TABLE 4
| Detection of | Sensitivity of the probe | Specificity of | AUC |
| Detection 1 | 0.987 | 0.997 | 0.992 |
| Detection 2 | 0.987 | 0.995 | 0.991 |
| Detection 3 | 0.887 | 0.992 | 0.942 |
| Detection 4 | 0.987 | 0.995 | 0.991 |
| Detection 5 | 0.987 | 0.995 | 0.991 |
| Detection 6 | 0.987 | 0.995 | 0.991 |
| Detection 7 | 0.717 | 0.995 | 0.856 |
| Detection 8 | 0.635 | 0.997 | 0.816 |
| Detection 9 | 0.692 | 0.995 | 0.843 |
As can be seen from table 4, based on the biomarkers provided by the present disclosure, accurate detection of mycobacterium avium can be achieved.
Comparative example 1
The moxC gene, the hldE gene, the ino 1G 832A site, the clpB G2124C site, the rrl G447A site, the rrl T455A site, the Rv 1461G 2133A site, the Rv 2402G 570C site, the rpsK C90G site and the pks 13G 672C site are respectively used as molecular markers, whether the sequences of the molecular markers exist in the total nucleic acid sequences of the mycobacteria obtained in the example 2 is judged, the mycobacteria containing the sequences of the molecular markers are judged to be mycobacterium avium, and the detection result is obtained.
For each detection result, ROC analysis was performed to determine the specificity, sensitivity, and AUC value of each detection, and the results are shown in table 5.
TABLE 5
As is clear from Table 5, the detection accuracy of each of the molecular markers mentioned in the present comparative examples against Mycobacterium avium is inferior to that of each of the molecular markers mentioned in the examples disclosed.
Comparative example 2
The Hsp65 gene sequences were determined by using base mutations at positions 184 (SNP1), 241 (SNP2), 299 (SNP3), and 346 (SNP4) from the 5' end as the detection sites in common, and the Hsp65 gene sequences in the total nucleic acid sequences of the respective mycobacteria obtained in example 2 were determined, and when the SNP1 was C, SNP2, G, SNP3, and T, SNP4 was C, the corresponding mycobacteria were determined to be mycobacterium avium, and the detection results were obtained.
ROC analysis was performed on the detection results to determine the specificity, sensitivity, and AUC values of the detection, and the results are shown in table 6.
TABLE 6
| Detection of | Sensitivity of the probe | Specificity of | AUC |
| Detection 20 | 0.503 | 0.995 | 0.749 |
As can be seen from Table 6, the detection sites referred to in the present comparative examples were less accurate in detection of M.avium than the respective molecular markers referred to in the disclosed examples.
The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure, as long as it does not depart from the spirit of the present disclosure.
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Claims (6)
1. A kit for detection of mycobacterium avium, comprising reagents for detecting a molecular marker, wherein the molecular marker comprises a nikA gene;
the reagent for detecting the molecular marker comprises a primer pair capable of specifically amplifying the molecular marker and/or a probe capable of specifically hybridizing with the molecular marker;
the primer pair comprises SEQ ID NO 1-2.
2. Use of a reagent for the detection of a molecular marker for the preparation of a kit for the detection of mycobacterium avium, characterized in that the molecular marker comprises the nikA gene;
the reagent for detecting the molecular marker comprises a primer pair capable of specifically amplifying the molecular marker and/or a probe capable of specifically hybridizing with the molecular marker;
the primer pair comprises SEQ ID NO 1-2.
3. A system for detection of mycobacterium avium, the system comprising sequencing means, computing means and output means;
the sequencing device is used for carrying out nucleic acid sequence sequencing on the total nucleic acid of the sample to be tested to obtain a total nucleic acid sequence;
the computing device includes a memory having a computer program stored therein and a processor configured to execute the computer program stored in the memory to effect the determination of:
if the total nucleic acid sequence contains nikA gene sequence, judging that the mycobacterium avium exists in the sample to be detected;
the output device is used for outputting the judgment result of the computing device.
4. The system of claim 3, further comprising a nucleic acid extraction device for performing nucleic acid extraction on the sample to be tested to obtain total nucleic acid of the sample to be tested.
5. The system of claim 4, wherein the nucleic acid extraction device comprises a nucleic acid extractor and/or a nucleic acid extraction kit.
6. The system of any one of claims 3 to 5, wherein the sequencing device comprises at least one of a Sanger sequencing platform, an Illumina Novaseq, a HiSeq Xten, and a HiSeq2500/2000/4000 sequencing platform.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10215238C1 (en) * | 2002-04-06 | 2003-08-14 | Cytonet Gmbh & Co Kg | Detecting mycobacteria and differentiating between Mycobacterium tuberculosis and Mycobacterium avium, comprises amplifying the 16S rRNA gene |
| CN105219882A (en) * | 2015-11-19 | 2016-01-06 | 北京利德曼生化股份有限公司 | Mycobacterium avium-intracellulare compound group detects by primer and probe and its detection method |
| CN107190087A (en) * | 2017-07-18 | 2017-09-22 | 武汉大学 | Kit and method based on the TaqMan non-binding mycobacterias of MGB probe in detecting people and combination mycobacteria |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7824858B2 (en) * | 2006-01-23 | 2010-11-02 | Quest Diagnostics Investments Incorporated | Assay for mycobacterium avium/intracellulare nucleic acid |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10215238C1 (en) * | 2002-04-06 | 2003-08-14 | Cytonet Gmbh & Co Kg | Detecting mycobacteria and differentiating between Mycobacterium tuberculosis and Mycobacterium avium, comprises amplifying the 16S rRNA gene |
| CN105219882A (en) * | 2015-11-19 | 2016-01-06 | 北京利德曼生化股份有限公司 | Mycobacterium avium-intracellulare compound group detects by primer and probe and its detection method |
| CN107190087A (en) * | 2017-07-18 | 2017-09-22 | 武汉大学 | Kit and method based on the TaqMan non-binding mycobacterias of MGB probe in detecting people and combination mycobacteria |
Non-Patent Citations (2)
| Title |
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| Genbank.Genbank Accession：CP046507.《Genbank》.2019, * |
| Jia et al..Combining comparative genomic analysis with machine learning reveals some promising diagnostic markers to identify five common pathogenic non-tuberculous mycobacteria.《Microbial Biotechnology》.2021, * |
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