CN112538538A - Kit and system for detecting mycobacterium abscessus - Google Patents
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Abstract
The kit comprises a reagent for detecting a molecular marker, wherein the molecular marker comprises at least one of a benM gene, an aqpZ gene, an aldHT gene, an osmX gene, a fsr gene, a rrl A1173G site and an aceE C867T site. By identifying the above molecular markers, the present disclosure enables accurate detection of mycobacterium abscessus.
Description
Technical Field
The present disclosure relates to the field of biotechnology, specifically, to a kit for mycobacterium abscessus detection, use of a reagent for detecting a molecular marker in preparation of a kit for mycobacterium abscessus detection, and a system for mycobacterium abscessus 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 abscessus.
In order to achieve the above objects, the present disclosure provides, in a first aspect, a kit for mycobacterium abscessus detection, comprising a reagent for detecting a molecular marker, wherein the molecular marker comprises at least one of a benM gene, an aqpZ gene, an aldHT gene, an osmX gene, a fsr gene, a rrl a1173G site, and an aceE C867T 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, SEQ ID NO 7-8, SEQ ID NO 9-10, SEQ ID NO 11-12 and SEQ ID NO 13-14.
In a second aspect, the present disclosure provides use of an agent for detecting a molecular marker including at least one of a benM gene, an aqpZ gene, an aldHT gene, an osmX gene, a fsr gene, a rrl a1173G site, and an aceE C867T site, in the preparation of a kit for mycobacterium abscessus 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, SEQ ID NO 7-8, SEQ ID NO 9-10, SEQ ID NO 11-12 and SEQ ID NO 13-14.
In a third aspect, the present disclosure provides a system for mycobacterium abscessus 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 a benM gene sequence, an aqpZ gene sequence, an aldHT gene sequence, an osmX gene sequence, a fsr gene sequence, a rrl A1173G site sequence and an aceE C867T site sequence, judging that the mycobacterium abscessus 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, HiSeq 2500/2000/4000 sequencing platform.
Through the technical scheme, the accurate detection of the mycobacterium abscessus can be at least partially realized by identifying molecular markers such as benM genes, aqpZ genes, aldHT genes, osmX genes, fsr genes, rrl A1173G sites or aceE C867T sites.
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 abscessus detection, the kit comprising reagents for detecting molecular markers, wherein the molecular markers comprise at least one of a benM gene, an aqpZ gene, an aldHT gene, an osmX gene, a fsr gene, a rrl a1173G site, and an aceE C867T site.
The inventor of the present disclosure finds that specific molecular markers different from other non-tuberculous mycobacteria or tuberculous mycobacteria exist in the mycobacterium abscesses, and are benM gene, aqpZ gene, aldHT gene, osmX gene, fsr gene, rrl A1173G site and aceE C867T site, and by detecting these molecular markers, the mycobacterium abscesses can be accurately distinguished from other non-tuberculous mycobacteria or tuberculous mycobacteria, thereby at least partially realizing the accurate detection of the mycobacterium abscesses.
Specifically, the gene numbers of the above-mentioned molecular markers are shown in Table 1.
TABLE 1
| Molecular marker | Gene numbering |
| benM Gene | 5962568 |
| aqpZ gene | 5963125 |
| aldHT gene | 5963355 |
| osmX gene | 5963752 |
| fsr gene | 5963862 |
| rrl A1173G site | 2700466 |
| aceE C867T site | 448824781 |
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, SEQ ID NO. 11-12, and SEQ ID NO. 13-14.
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 an agent for detecting a molecular marker including at least one of a benM gene, an aqpZ gene, an aldHT gene, an osmX gene, a fsr gene, a rrl a1173G site, and an aceE C867T site, in preparing a kit for mycobacterium abscessus detection.
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, SEQ ID NO 11-12 and SEQ ID NO 13-14.
A third aspect of the present disclosure provides a system for mycobacterium abscessus 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 a benM gene sequence, an aqpZ gene sequence, an aldHT gene sequence, an osmX gene sequence, a fsr gene sequence, a rrl A1173G site sequence and an aceE C867T site sequence, judging that the mycobacterium abscessus 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, HiSeq 2500/2000/4000 sequencing platform.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
The starting materials, reagents, instruments and equipment referred to in the examples of the present disclosure may be obtained by purchase, 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.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 genomic analysis on the genome data of the discovery group, and determining distinguishing genes and distinguishing SNPs sites which are different from the genomes of other NTM strains in the genomes of 36 strains of mycobacterium abscessus, wherein the distinguishing genes and the distinguishing SNPs sites are respectively benM genes, aqpZ genes, aldHT genes, osmX genes, fsr genes, badR genes, yxaF genes, ohrR genes, rrl A1173G sites, aceE C867T sites and Rv1248C C1647G sites.
Aiming at the distinguishing genes and the distinguishing SNPs sites, a chi 2 test and a 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 the mycobacterium abscessus are screened from the distinguishing genes and the distinguishing SNPs sites, wherein the specific genes and the specific SNPs sites are respectively benM genes, aqpZ genes, aldHT genes, osmX genes, fsr genes, rrl A1173G sites and aceE C867T sites, and the specific genes and the specific SNPs sites are used as biomarkers for detecting the mycobacterium abscessus.
Example 2
This example is used to verify the detection effect of the biomarkers provided by the present disclosure on mycobacterium abscessus.
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 as mycobacterium abscessus, 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 detection 1-10, 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 | 0.909 | 1.000 | 0.955 |
| Detection 2 | 0.909 | 0.996 | 0.952 |
| Detection 3 | 0.846 | 0.996 | 0.922 |
| Detection 4 | 0.909 | 0.996 | 0.952 |
| Detection 5 | 0.909 | 0.996 | 0.952 |
| Detection 6 | 0.909 | 0.996 | 0.952 |
| Detection 7 | 0.909 | 0.996 | 0.952 |
| Detection 8 | 0.909 | 0.996 | 0.952 |
| Detection 9 | 0.839 | 0.996 | 0.917 |
| Test 10 | 0.821 | 1.000 | 0.911 |
As can be seen from table 4, based on the biomarkers provided by the present disclosure, accurate detection of mycobacterium abscessus can be achieved.
Comparative example 1
The presence or absence of the sequences of each molecular marker in the total nucleic acid sequences of each mycobacterium obtained in example 2 was determined using the badR gene, the ohrR _1 gene, the yxaF _2 gene, and the Rv1248C C1647G site as molecular markers, and the mycobacterium containing the sequences of each molecular marker was determined to be mycobacterium abscessus, and the detection result was 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 abscessus is inferior to that of each of the molecular markers mentioned in the disclosed examples.
Comparative example 2
The 130 th (SNP1), 154 th (SNP2), 181 th (SNP3), 184 th (SNP4), 193 th (SNP5), 299 th (SNP6) and 346 th (SNP7) base mutations in the sequence of Hsp65 gene were used together as detection sites, and the Hsp65 gene sequence in the total nucleic acid sequence of each mycobacterium obtained in example 2 was determined, and when SNP1 was T, SNP2, G, SNP3 was T, SNP4, T, SNP5 was C, SNP6 was T, A, SNP7 was T, the corresponding mycobacterium was determined to be mycobacterium abscessus, and the detection result was 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 15 | 0.070 | 1.000 | 0.465 |
As can be seen from Table 6, the detection sites referred to in the present comparative examples were less accurate in detection of Mycobacterium abscessus 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 (10)
1. The kit for detecting the mycobacterium abscesses is characterized by comprising a reagent for detecting a molecular marker, wherein the molecular marker comprises at least one of a benM gene, an aqpZ gene, an aldHT gene, an osmX gene, a fsr gene, a rrl A1173G site and an aceE C867T site.
2. The kit according to claim 1, wherein 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.
3. The kit according to claim 2, wherein 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, SEQ ID NO 7-8, SEQ ID NO 9-10, SEQ ID NO 11-12 and SEQ ID NO 13-14.
4. Use of a reagent for detecting a molecular marker comprising at least one of a benM gene, an aqpZ gene, an aldHT gene, an osmX gene, a fsr gene, a rrlA1173G site, and an aceE C867T site in the preparation of a kit for detecting mycobacterium abscessus.
5. The use according to claim 4, wherein 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.
6. The use of claim 5, wherein 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, SEQ ID NO 7-8, SEQ ID NO 9-10, SEQ ID NO 11-12 and SEQ ID NO 13-14.
7. A system for mycobacterium abscessus 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 a benM gene sequence, an aqpZ gene sequence, an aldHT gene sequence, an osmX gene sequence, a fsr gene sequence, a rrlA1173G site sequence and an aceE C867T site sequence, judging that the mycobacterium abscessus exists in the sample to be detected;
the output device is used for outputting the judgment result of the computing device.
8. The system of claim 7, further comprising a nucleic acid extraction device for performing nucleic acid extraction on the sample to be tested to obtain total nucleic acids of the sample to be tested.
9. The system of claim 8, wherein the nucleic acid extraction device comprises a nucleic acid extractor and/or a nucleic acid extraction kit.
10. The system of any one of claims 7 to 9, wherein the sequencing device comprises at least one of a Sanger sequencing platform, Illumina Novaseq, HiSeq Xten, HiSeq 2500/2000/4000 sequencing platform.
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