CN112538542B - Kit and system for detecting mycobacterium cheloni - Google Patents
Kit and system for detecting mycobacterium cheloni Download PDFInfo
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- CN112538542B CN112538542B CN202011356499.1A CN202011356499A CN112538542B CN 112538542 B CN112538542 B CN 112538542B CN 202011356499 A CN202011356499 A CN 202011356499A CN 112538542 B CN112538542 B CN 112538542B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
- G16B20/30—Detection of binding sites or motifs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The present disclosure relates to a kit and a system for mycobacterium cheloni detection, the kit comprising a reagent for detecting a molecular marker, wherein the molecular marker comprises at least one of yjcH gene, mpr gene, rrl C2638T site and rrl G2654A site. By identifying the above molecular markers, the present disclosure enables accurate detection of mycobacterium cheloni.
Description
Technical Field
The present disclosure relates to the field of biotechnology, specifically to a kit for mycobacterium cheloniae detection, use of a reagent for detecting a molecular marker in preparation of a kit for mycobacterium cheloniae detection, and a system for mycobacterium cheloniae detection.
Background
Nontuberculous Mycobacterium (NTM) refers to a Mycobacterium other than Mycobacterium tuberculosis complex (Mtb) and Mycobacterium leprae, and is widely present in the natural environment. 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 increasing 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 (hsp 65), 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 the NTM species with close genetic relationship cannot be accurately distinguished.
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 cheloni.
In order to achieve the above objects, the present disclosure provides, in a first aspect, a kit for mycobacterium cheloni detection, the kit comprising reagents for detecting a molecular marker, wherein the molecular marker comprises at least one of yjcH gene, mpr gene, rrl C2638T site, and rrl G2654A 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 in SEQ ID NO 1-2, SEQ ID NO 3-4, SEQ ID NO 5-6 and SEQ ID NO 7-8.
In a second aspect, the present disclosure provides use of a reagent for detecting a molecular marker comprising at least one of yjcH gene, mpr gene, rrl C2638T site and rrl G2654A site in the preparation of a kit for mycobacterium chelonii detection.
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 in SEQ ID NO 1-2, SEQ ID NO 3-4, SEQ ID NO 5-6 and SEQ ID NO 7-8.
In a third aspect, the present disclosure provides a system for mycobacterium cheloniae 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 yjcH gene sequence, mpr gene sequence, rrl C2638T site sequence and rrl G2654A site sequence, judging that mycobacterium cheloni 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, where the nucleic acid extraction device is configured to extract nucleic acid from 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 mycobacterium cheloni can be at least partially realized by identifying molecular markers such as yjcH genes, mpr genes, rrl C2638T sites or rrl G2654A sites.
Additional features and advantages of the present 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 cheloni detection, the kit comprising reagents for detecting a molecular marker, wherein the molecular marker comprises at least one of a yjcH gene, an mpr gene, a rrl C2638T site, and a rrl G2654A site.
The inventor of the present disclosure finds that, specific molecular markers different from other non-tuberculous mycobacteria or tubercle mycobacteria exist in mycobacterium cheloni, namely yjcH gene, mpr gene, rrl C2638T site and rrl G2654A site, and by detecting the molecular markers, mycobacterium cheloni can be accurately distinguished from other non-tuberculous mycobacteria or tubercle mycobacteria, so as to at least partially realize the accurate detection of mycobacterium cheloni.
Specifically, the gene numbers of the above-mentioned molecular markers are shown in Table 1.
TABLE 1
| Molecular marker | Gene numbering |
| yjcH gene | 31678822 |
| mpr gene | 31681477 |
| rrl C2638T site | 2700466 |
| rrl G2654A site | 2700466 |
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 in SEQ ID NOS 1 to 2, 3 to 4, 5 to 6, and 7 to 8.
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 yjcH gene, mpr gene, rrl C2638T site and rrl G2654A site in the preparation of a kit for mycobacterium cheloni detection.
Alternatively, the reagent for detecting a molecular marker may comprise 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 and SEQ ID NO. 7-8.
A third aspect of the present disclosure provides a system for mycobacterium cheloni 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 yjcH gene sequence, mpr gene sequence, rrl C2638T site sequence and rrl G2654A site sequence, judging that mycobacterium cheloni 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 apparatus may be, for example, a second generation sequencing apparatus, for example, the sequencing apparatus 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 method of biomarker determination provided by the present disclosure.
81 NTM strains having a complete genome were collected as discovery groups including 7 M.kansasii, 27 M.avium, 6 M.intracellulare, 5 M.cheloniae 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 genome data of the found groups, and determining distinguishing genes and distinguishing SNPs sites which are different from genomes of other NTM strains in the genomes of the 5 mycobacterium cheloni, wherein the distinguishing genes are yjcH genes, mpr genes, yaeQ genes, copB genes, pglH genes, rebH genes, rrl C2638T sites, rrl G2654A sites and rrl A2603G sites.
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 genomes of other NTM strains in the genome of mycobacterium cheloniae are screened out from the distinguishing genes and the distinguishing SNPs sites, wherein the specific genes and the specific SNPs sites are yjcH genes, mpr genes, rrl C2638T sites and rrl G2654A sites respectively, and the specific genes and the specific SNPs sites are used as biomarkers for detecting the mycobacterium cheloniae.
Example 2
This example is used to verify the detection effect of the biomarkers provided by the present disclosure on mycobacterium cheloni.
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 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 cheloniae, and the detection result is obtained. The molecular markers targeted for each detection are shown in table 3.
TABLE 3
| Detection of | Molecular marker |
| Detection 1 | yjcH gene + mpr gene + rrl C2638T site + rrl G2654A site |
| Detection 2 | yjcH gene+ mpr Gene |
| Detection 3 | rrl C2638T site + rrl G2654A site |
| Detection 4 | yjcH gene |
| Detection 5 | Mpr gene |
| Detection 6 | rrl C2638T site |
| Detection 7 | rrl G2654A site |
For the detection results of detections 1 to 7, ROC analysis was performed to determine the specificity, sensitivity, and AUC value of each detection, and the results are shown in table 4.
TABLE 4
| Detection | Sensitivity of the probe | Specificity of the drug | AUC |
| Detection 1 | 0.957 | 0.958 | 0.958 |
| Detection 2 | 0.957 | 0.952 | 0.955 |
| Detection 3 | 0.553 | 0.958 | 0.756 |
| Detection 4 | 0.957 | 0.952 | 0.955 |
| Detection 5 | 0.957 | 0.952 | 0.953 |
| Detection 6 | 0.511 | 0.958 | 0.734 |
| Detection 7 | 0.553 | 0.958 | 0.756 |
As can be seen from table 4, based on the biomarkers provided by the present disclosure, accurate detection of mycobacterium cheloni can be achieved.
Comparative example
The yaeQ gene, copB gene, pglH gene, rebH gene and rrl A2603G site were used as molecular markers, and the presence or absence of the sequences of the molecular markers in the total nucleic acid sequences of the mycobacteria obtained in example 2 was determined, and the mycobacteria containing the sequences of the molecular markers were determined to be Mycobacterium cheloni, and the results of the detection were 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
| Detection of | Molecular marker | Sensitivity of the probe | Specificity of | AUC |
| Detection 8 | yaeQ gene | 0.830 | 0.620 | 0.725 |
| Detection 9 | CopB Gene | 0.851 | 0.952 | 0.901 |
| Test 10 | pglH gene | 0.787 | 0.952 | 0.869 |
| Detection 11 | rebH gene | 0.702 | 0.952 | 0.820 |
| Detection 12 | rrl A2603G site | 0.362 | 0.966 | 0.604 |
As is clear from Table 5, the detection accuracy of each of the molecular markers mentioned in the present comparative examples against Mycobacterium cheloni was inferior to that of each of the molecular markers mentioned in the disclosed examples.
The preferred embodiments of the present disclosure have been described above in detail, 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 belong to 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 gist of the present disclosure.
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Claims (6)
1. A kit for the detection of mycobacterium cheloni, comprising reagents for detecting a molecular marker, wherein the molecular marker comprises a yjcH gene; the gene number of the yjcH gene is 31678822;
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 detecting a molecular marker in the preparation of a kit for mycobacterium cheloniae detection, wherein the molecular marker comprises the yjcH gene; the gene number of the yjcH gene is 31678822;
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 mycobacterium cheloni 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 a yjcH gene sequence, judging that mycobacterium cheloni exists in the sample to be detected; the gene number of the yjcH gene is 31678822;
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, illumina Novaseq, hiSeqXten, hiSeq2500/2000/4000 sequencing platform.
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| CA2119188A1 (en) * | 1993-04-05 | 1994-10-06 | Patricia A. Spears | Detection and identification of mycobacteria |
| US8029806B2 (en) * | 2008-04-16 | 2011-10-04 | Asiagen Corporation | Method for diagnosing of Mycobacterium tuberculosis |
| CN104164475B (en) * | 2013-05-17 | 2017-05-03 | 北京市结核病胸部肿瘤研究所 | Identification method, kit and universal primer pair for mycobacterium, and application of rpsA gene |
| CN109468316B (en) * | 2018-12-10 | 2020-06-05 | 上海市肺科医院 | Gene sequence composition and application thereof in preparation of mycobacterium lung disease detection kit |
| CN110656193A (en) * | 2019-11-22 | 2020-01-07 | 天津市海河医院 | Membrane strip for identifying mycobacterium tuberculosis, mycobacterium cheloniae and mycobacterium abscessus and preparation and use methods |
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