WO2019206172A1 - Mycobacterium tuberculosis h37rv encoding gene and application thereof - Google Patents

Abstract

Provided are a mycobacterium tuberculosis H37Rv encoding gene and an application thereof, specifically a new missing annotated encoding gene Rv3128(+|3493573-3493707|), which may be used as a standard gene for mycobacterium tuberculosis complex molecular identification, and which may be used for mycobacterium tuberculosis complex molecular identification and clinical detection.

Description

Mycobacterium tuberculosis H37Rv coding gene and its application Technical field

The invention relates to the field of gene detection, in particular to the identification of pathogenic species.

Background technique

Mycobacterium tuberculosis (MTB) is a pathogen that causes tuberculosis in humans. It can invade all organs of the body, but tuberculosis is the most common. Tuberculosis is an infectious disease that is extremely important to date and poses a serious threat to human health. According to the WHO, about 8 million new cases occur each year, and at least 3 million people die from the disease. The clinical strain of MTB is difficult to culture, grows slowly, can be cross-infected with other mycobacteria, and the symptoms of tuberculosis and other respiratory infections are difficult to distinguish, which brings great difficulties to clinical rapid diagnosis and treatment. Therefore, the establishment of rapid, accurate, specific, sensitive and inexpensive methods for detecting tuberculosis is a necessary prerequisite for effective treatment and control of the spread of tuberculosis, and it is also a new challenge and new task for the detection of mycobacteria in clinical laboratories.

Mycobacterium tuberculosis complex (MTBC), including M. tuberculosis, M. africanum, M. orgis, M. bovis, M. microti, M. canettii, M. caprae, M. pinnipedii, M. suricattae Mycobacteria, such as M.mungi, which cause tuberculosis in humans and other living organisms. At present, the identification methods of MTBC at home and abroad are mainly divided into the following three categories: traditional isolation and culture methods; molecular level detection (IS6110, restriction fragment length polymorphism analysis, multi-locus variable number repeat fragment polymorphism analysis, etc.); Chromatographic analysis of bacterial components (fatty acids, mycolic acids). Although the three types of methods have their own advantages, they also have some shortcomings, such as the long period of traditional isolation and culture and the low culturability of the cells; the molecular level detection is still poor in specificity, sensitivity and simplicity; The analysis of the composition characteristics is costly and complicated.

MTB H37Rv completed genome-wide sequencing in 1998 and was the first MTB strain to complete full-sequence sequencing. Since then, researchers in various countries have been perfecting and supplementing the H37Rv gene annotation database based on algorithms such as algorithm optimization, annotation software update, transcriptomics and proteomics. However, since MTB belongs to prokaryotes, it is possible in genome annotation due to the lack of prokaryotic genome annotation technology itself.

There are annotation errors (over-annotation, gene boundary errors and ORF initiation, termination site errors, alternative splicing, ribosome translocation, and leak annotation), which plague deep and accurate analytical biological mechanisms. In order to solve this problem, Protein Genomics (Proteogenomics) has been used for the correction of H37Rv annotated genes. However, high proportion of false positives, conventional techniques are difficult to predict gene prediction, new gene verification, new gene function analysis and its applications. It is a difficult problem in this field.

In general, the traditional M. tuberculosis complex (MTBC) identification strategy has the defects of long cycle, cumbersome steps, low specificity and sensitivity. In order to further re-examine the H37Rv genome, we found that the missing annotation gene in H37Rv ensures that the H37Rv whole genome missing annotation gene and its application technology in MTBC molecular identification are effectively protected, and the new H37Rv gene is rapidly and accurately identified in the MTBC group. The method is imperative.

Summary of the invention

An object of the present invention is to provide a novel coding gene for Mycobacterium tuberculosis H37Rv, which is a H37Rv leak-annotation coding gene Rv3128 (+|3493573-3493707|), which can be used as a barcode molecular marker of Mycobacterium tuberculosis complex group For detecting a Mycobacterium tuberculosis complex, the sequence of which is shown in SEQ ID NO.

Other objects of the present invention include providing a specific PCR primer that can be used to amplify the above-described coding gene and providing a detection method for detecting or identifying the presence or absence of a mycobacterial complex in a sample; the present invention also provides a gene associated with the above-described coding gene. Detection kit and application of the above genes.

According to one aspect of the present invention, by comparing protein genomics research techniques, a protein coding sequence in H37Rv that is difficult to be discovered by gene prediction software is found, which is effective in distinguishing MTBC from other species belonging to the same genus. The gene is a missing annotation gene of Mycobacterium tuberculosis H37Rv, which is Rv3128 (+|3493573-3493707|). After NCBI-BLASTP, there is no alignment in the database, and it belongs to a functionally unknown protein. The comparative genomics study found that the gene sequence can distinguish the Mycobacterium tuberculosis complex (MTBC) strain from other species of Mycobacterium.

Specifically, a primer for the specific amplification of the Rv3128 (+|3493573-3493707|) gene of MTBC is designed, which is a primer proposed in the present invention, and the primer sequence is:

F: 5'-TTGAGGCTGTCTCGGTAGGT-3';

R: 5'-GGGATAACGGGTTTAGATTTCG-3'.

According to the presence or absence of the PCR product of the gene DNA sequence in the sample to be tested or the difference in DNA sequence, MTBC can be quickly and accurately identified.

According to another aspect of the present invention, the present invention specifically establishes a method for detecting or identifying a Mycobacterium tuberculosis complex based on the above novel standard coding gene of M. tuberculosis H37Rv, the steps are as follows:

(1) separating and extracting genomic DNA from the sample to be tested;

(2) Using the DNA obtained in the step (1) as a template, PCR amplification was carried out using the following primers:

F: 5'-TTGAGGCTGTCTCGGTAGGT-3' (SEQ ID NO. 4);

R: 5'-GGGATAACGGGTTTAGATTTCG-3' (SEQ ID NO. 5).

(3) performing gel electrophoresis analysis or sequencing on the DNA product amplified in step (2);

(4) The result of the step (3) is compared with the barcode gene Rv3128 (+|3493573-3493707|), and if the homology is greater than 99%, it is determined that the sample to be tested contains the Mycobacterium tuberculosis complex.

Further, the above detection method, according to the principle of DNA barcode, preliminary electrophoresis analysis of the PCR product, if the strain to be tested does not have a target band, indicating that the strain is not MTBC; if there is a band, further sequencing verification, sequencing will be sequenced Homologous comparison and alignment with the standard sequence of R373 (+|3493573-3493707|) of H37Rv, to obtain similarity between sequences, if the sequence homology is greater than 99%, it can be determined that the strain may be MTBC; The DNA barcode sequence of the strain was identified and the standard sequence clustering was used to distinguish the MTBC family from non-tuberculous mycobacteria, common respiratory pathogens and common respiratory viruses.

The detection method can be used for the identification of strains of Mycobacterium tuberculosis complex group, and can also be used for clinical rapid test. The sample to be tested may be H37Rv strain, other MTBC, non-tuberculous mycobacteria, common respiratory pathogens, common viral strains of the respiratory tract; or direct use of sputum, saliva or blood of tuberculosis and other respiratory patients.

Based on the above method, the present invention also provides a detection kit, which is provided with a reagent for detecting a new standard coding gene of Mycobacterium tuberculosis H37Rv, and at the same time, may be provided by a government drug regulatory agency, Information on the manufacture, use and sale of pharmaceuticals or biological products. For example, after PCR amplification, the reagent for directly detecting the Rv3128 (+|3493573-3493707|) gene in the sample, for example, may contain an amplification primer, dNTP, a DNA polymerase for PCR reaction, a buffer thereof, and a digestion reaction. And/or one or more of the reagents required for sequencing the reaction, and the like. It is known to those skilled in the art that the above components are only illustrative, for example, the primers may employ the above specific PCR primers, and the DNA polymerase used for the PCR reaction is an enzyme that can be used for PCR amplification. Detection of the coding genes of the invention can also be provided in an integrated manner, such as a gene chip.

Advantageous Effects: The present invention provides a standard gene and molecular identification method for molecular identification of Mycobacterium tuberculosis complex (MTBC), which can effectively distinguish MTBC from other species belonging to the same genus. The identification method of the gene overcomes the shortcomings of the primer design and the poor repeatability of the existing Mycobacterium tuberculosis complex group, and has the characteristics of universal, easy amplification and easy comparison, and can accurately classify the group. It is identified from other mycobacteria or other respiratory infections with close kinship, providing powerful technical means and research tools for tuberculosis epidemiology investigation and rapid diagnosis and identification of clinical tuberculosis patients.

DRAWINGS

Figure 1: Peptide mapping matching evidence supporting the discovery of new coding genes;

Figure 2: Comparison of the mass spectrum of the synthetic peptide with the original identified peptide mass spectrum;

Figure 3: Corresponding diagram of the protein sequence encoded by the ORF in the region of the peptide; the underlined portion is a peptide that is proteomically identified and verified by the synthetic peptide;

Figure 4: Rv3128 (+|3493573-3493707|) standard gene sequence homology comparison;

Figure 5: BLASTP results of the protein sequence corresponding to the H37Rv strain Rv3128 (+|3493573-3493707|) gene;

Figure 6: Results of agarose gel electrophoresis of PCR amplification products of Rv3128(+|3493573-3493707|) specific primers;

Among them, the specific information of each lane sample is shown in Table 1.

Figure 7: Comparison of PCR amplification sequencing results of Rv3128 (+|3493573-3493707|) gene with standard sequences.

detailed description

The invention is further illustrated by the following specific examples, without limiting the scope of the claims of the invention. The reagents used in the present invention are all commercially available.

Example 1: Search for the leak-annotated coding gene of the H37Rv strain genome

1.1 High coverage proteome validation of the H37Rv strain genome

The deep coverage of the proteome was performed on the H37Rv strain using the high-coverage proteome technique. Based on the Tuberculosis (20160307) database, the genome was annotated with the pFind 3 engine for gene validation. In order to discover the new protein coding region, we used the pAnno software to perform a six-frame database translation of H37Rv's genome-wide (NC_000962.3) file published by NCBI using pAnno software, and used this database to perform new peptides on mass spectrometry data. Identification of segments and new proteins. In order to reduce the false positive rate, we used three filtering methods for estimating the class FDR separately for the annotated peptide and the new peptide in the data filtering process, namely S-FDR, T-FDR I and T-FDR II.

Through data analysis, we identified 3238 H37Rv annotated genes with coverage of more than 80% of the strain, which is the largest H37Rv protein profile data reported so far. In addition, after three kinds of FDR≤1 filtration, we obtained new peptides. In order to further ensure the quality of the new peptides, we performed a spectral quality screening on the spectra corresponding to the remaining new peptides, and finally retained some peptides with good spectral quality. To further investigate the higher quality of these peptides due to the single amino acid mutation in the annotated peptide, we performed an amino acid mutation check to ensure that these new peptides are newly identified for H37Rv.

1.2 Encoding protein and database validation of Rv3128(+|3493573-3493707|) gene

After high-coverage proteomic validation, we found some suspected new leak-reported peptides, and performed peptide synthesis verification on the above-mentioned highly reliable suspected new peptides. The scores of the new peptide original spectrum and peptide synthesis spectrum were scored. ≥0.9 As the similarity threshold, after scoring screening, one peptide was verified, corresponding to the open reading frame (ORF), which is the potential leaky annotation gene of the current H37Rv strain.

Among them, we found that the new leaky annotation gene Rv3128 (+|3493573-3493707|), compared with BLASTP, did not match any sequence and belonged to a functionally unknown protein. We detected the peptide AGEVDTVVAGPADDR (SEQ ID NO. 6) and corresponded to the new gene Rv3128 (+|3493573-3493707|). As shown in Figure 1, the spectrum quality is very good, b/y ions are continuously matched, and the peaks are mixed. The signal is low and the results are very reliable.

To further confirm this identification, we chemically synthesized the peptide according to the amino acid sequence of our newly identified peptide and generated a secondary spectrum of the synthetic peptide using the mass spectrometric conditions described above.

We verified the high-energy collision MS2 produced by the synthetic peptides. The primary and secondary daughter ions were in agreement with the theoretical values, indicating that the peptide sequence we synthesized was correct; on this basis, we manually checked the scale according to the large scale. The MS2 of the synthetic peptides identified by the proteomic data and the large-scale identification of the new peptides were almost identical. The cosin value obtained by the similarity of the daughter ions was 0.95, which proved that we identified from H37Rv. The new peptide is correct. (figure 2).

After confirming the sequence of the above-mentioned leak-inscribed peptide segment, according to the position of the gene in which the above-mentioned peptide segment is located, the region including the previous stop codon and the latter stop codon is bounded, and an open reading frame containing the above-mentioned new leak-inscribed peptide segment is obtained ( ORF) DNA sequence as shown in SEQ ID NO.

The correspondence between the open reading frame coding and the amino acid sequence is shown in FIG.

Further translation verification revealed that the actual gene sequence (SEQ ID NO. 1) was 135 bp from the GTG in the open reading frame DNA (SEQ ID NO. 2), encoding 44 amino acids with a theoretical molecular weight of 4.48 kDa. Rv3128 (+|3493573-3493707|) gene.

The gene theory encodes the amino acid sequence of the product as shown in SEQ ID NO. 3:

The amino acid sequence of the product encoded by the theoretical gene shown in SEQ ID NO. 3 was subjected to NCBI-BLASTP analysis, and the database did not have any similar sequence and belonged to a functionally unknown protein. (See Figure 5). This indicates that the Rv3128 (+|3493573-3493707|) gene product we detected was omitted from the H37Rv strain database.

We performed a comparative genomic local BLAST analysis of the DNA sequence of the Rv3128 (+|3493573-3493707|) gene, as shown in Figure 5. The results indicated that the Rv3128(+|3493573-3493707|) gene sequence belongs to the MTBC family-specific gene. There are no homologous sequences in other species, which indicates that the Rv3128 (+|3493573-3493707|) gene sequence we found in the H37Rv strain has good sequence specificity, and can bind MTBC to other mycobacteria in the same genus. It is distinguished from other respiratory infections.

Example 2: Establishing a method for identifying MTBC complexes

(1) Design primers:

Based on the CDS sequence of the Rv3128 (+|3493573-3493707|) gene shown in SEQ ID NO. 1, PCR primers were designed using Oligo7.0. The primer sequences are as follows:

F: 5'-TTGAGGCTGTCTCGGTAGGT-3' (SEQ ID NO. 4);

R: 5'-GGGATAACGGGTTTAGATTTCG-3' (SEQ ID NO. 5)

The positional relationship between the above primers and the Rv3128 (+|3493573-3493707|) gene is as follows, wherein the primers correspond to positional subscripts, and the Rv3128 gene sequence is a gray background region.

(2) Extracting the total DNA of the strain to be tested including M. tuberculosis H37Rv, 40 strains of Mycobacterium standard strains were preserved by the Chinese Medical Bacterial Culture Collection and Management Center (CMCC), and the remaining 16 non-tuberculous mycobacteria were The clinical isolates of the 309 Hospital of the Chinese People's Liberation Army have completed the sequencing, alignment and NCBI sequence submission of the 16S RNA gene of the strains. The strains to be tested are shown in Table 1:

Table 1. Selected strains selected

(3) The DNA fragment was amplified, subjected to polymerase chain reaction (PCR) reaction, and amplified using the above F/R primer.

The PCR system (25 μL) was dd H ₂ O (9.5 μL), 2XTaq PCR MasterMix (TIANGEN, 12.5 μL) primer F (10 μM, 1 μL), primer R (10 μM, 1 μL), DNA template (1 μL);

The amplification procedure: pre-denaturation at 94 ° C for 3 min, denaturation at 94 ° C for 30 s, annealing at 58 ° C for 30 s, extension at 72 ° C for 1 min, 35 cycles, and extension at 72 ° C for 5 min.

(4) The amplified product was electrophoresed and detected by electrophoresis in agarose gel and 1×TBE electrophoresis solution. As a result, as shown in Fig. 6, the MTBC and the positive control group showed an amplified band at 407 bp, and the amplification result was in agreement with the expectation, and the specificity was 98.3%.

(5) In order to further verify the sequence of the amplified DNA, we sequenced the amplified sequence and compared it with the leaked annotation sequence, as shown in Figure 7. The results were exactly as expected, and the sequence was correct, which further verified the new leak. Annotate the presence of genes.

This indicates that the method based on Rv3128 (+|3493573-3493707|) gene for MTBC complex identification is true and reliable.

Claims (10)

1. A Mycobacterium tuberculosis H37Rv encoding gene Rv3128 (+|3493573-3493707|), the nucleotide sequence of which is shown in SEQ ID NO.
2. The Mycobacterium tuberculosis H37Rv encoding gene Rv3128 (+|3493573-3493707|) according to claim 1, characterized in that the gene encodes an amino acid as shown in the sequence of SEQ ID NO.
3. A barcode molecular marker for use in detecting and/or identifying a Mycobacterium tuberculosis complex comprising the Mycobacterium tuberculosis H37Rv encoding gene Rv3128 (+|3493573-3493707|) according to claim 1 as a standard detection gene.
4. A specific PCR primer for amplifying the Mycobacterium tuberculosis H37Rv encoding gene Rv3128 (+|3493573-3493707|) according to claim 1.
5. The PCR primer of claim 4, wherein the sequence of the primer is:

   F: 5'-TTGAGGCTGTCTCGGTAGGT-3';

   R: 5'-GGGATAACGGGTTTAGATTTCG-3'.
6. A method for detecting the identification of a Mycobacterium tuberculosis complex comprises the following steps:

   (1) separating and extracting genomic DNA from the sample to be tested;

   (2) using the DNA obtained in the step (1) as a template, adding an amplification primer, and performing a polymerase chain reaction;

   (3) performing gel electrophoresis analysis or sequencing on the DNA product amplified in step (2);

   (4) Aligning the result of the step (3) with the Rv3128 (+|3493573-3493707|) as a standard detection gene according to claim 1, and determining whether or not the tuberculosis branch is present in the sample to be tested based on the homology thereof Bacillus complex.
7. The detection method according to claim 6, wherein the amplification primer sequence described in the step (2) is:

   F: 5'-TTGAGGCTGTCTCGGTAGGT-3';

   R: 5'-GGGATAACGGGTTTAGATTTCG-3'.
8. The detecting method according to claim 6, wherein in the step (4), if the homology is greater than 99%, it is determined that the sample to be tested contains the Mycobacterium tuberculosis complex.
9. A detection kit comprising the Mycobacterium tuberculosis H37Rv encoding gene Rv3128 (+|3493573-3493707|) according to claim 1 and/or the specific PCR primer of claim 4.
10. Use of the Mycobacterium tuberculosis H37Rv encoding gene Rv3128 (+|3493573-3493707|) according to claim 1 for tuberculosis epidemiological investigation and/or rapid diagnosis and identification of clinical tuberculosis patients.

Images