CN114959074B - Specific sequence of clostridium difficile with high toxigenic and application thereof in identification and typing - Google Patents
Specific sequence of clostridium difficile with high toxigenic and application thereof in identification and typing Download PDFInfo
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Abstract
The invention discloses a novel specific sequence for high-yield clostridium difficile, and simultaneously discloses a real-time fluorescence quantitative PCR detection method for typing clostridium difficile based on the specific sequence, and a related primer pair and a probe for realizing detection of the high-yield clostridium difficile.
Description
Technical Field
The invention relates to a specific sequence of high-toxigenic clostridium difficile and application thereof in identification and typing.
Background
Clostridium difficile @Clostridioides difficileCD) is a gram-positive, obligate anaerobe capable of producing spores. CD is a major pathogen of Antibiotic-associated diarrhea (AAD) in hospitals, and can cause diarrhea of light to severe nature, colitis, pseudomembranous enteritis, toxic megacolon, etc., and severe cases can even lead to death.
In recent years, the incidence and mortality rate of clostridium difficile infection (Clostridiodes difficile infection, CDI) at home and abroad are increasing, causing tens of thousands of deaths annually, and the treatment cost is billions of dollars. In 2013 and 2019, us CDC (Center for Disease Control and Prevention) listed clostridium difficile infection as an urgent grade pathogen for "microbial causing public health threat". Clostridium difficile pathogenesis is largely attributed to these two toxins, enterotoxin a (TcdA) and cytotoxin B (TcdB). The main pathogenesis of Clostridium Difficile Infection (CDI) is that the clostridium difficile enters a human body through a spore to be planted in colon through a faecal-oral way, when intestinal flora is unbalanced due to long-term use of broad-spectrum antibiotics or proton pump inhibitors and the like, clostridium difficile is greatly propagated to generate TcdA and TcdB, and the cytoskeleton of the intestinal canal is destroyed, so that cell permeability is increased, and finally diarrhea and other disease symptoms are caused. In addition, in recent years, clostridium difficile high-yield strains NAP1/RT027/B1/ST1 and RT078/ST11 are outbreak in a plurality of countries in Europe and America, and not only TcdA and TcdB but also binary toxins (cdtA and cdtB) can be generated, so that the incidence rate and the death rate of CDI are greatly increased, and the wide attention of the whole society to CDI is brought about.
The rapid and accurate diagnosis of CDI is critical to guiding therapy and preventing the spread of pathogenic bacteria. Currently, there are a variety of laboratory detection methods for CDI for different detection targets. Methods for detecting free toxins in feces include Enzyme Immunoassay (EIA) and feces cytotoxicity assay (CTA). Measurement of specific antigen of clostridium difficile the EIA method measures Glutamate Dehydrogenase (GDH); methods for detecting strains of clostridium difficile include clostridium difficile cultivation (TC) and nucleic acid amplification assay (NAAT). Wherein, fecal CTA and TC are gold standards for detecting toxins or toxic strains respectively, but due to technical problems and overlong detection time, both methods are not conventionally used. The NAAT method is clostridium difficile detection based on a real-time fluorescence PCR (polymerase chain reaction) technology, a loop-mediated isothermal amplification technology or a microarray technology and other molecular detection technologies. NAATs detected a number of target genes including the tcdB, tcdA, and tcdC genes (117 as a marker for the 027 epidemic strain) or the binary toxin (cdt) gene. The NAATs method is very sensitive (average sensitivity is 96% (95% CI=0.93-0.98)), with a higher negative predictive value. Moreover, the detection speed is high, and the result can be obtained within one hour, so that the detection speed is often used as a CDI screening test. However, NAATs methods still have potential problems, such as the possibility of false negatives due to genetic variations, tcdB or tcdA gene fragments. The reliability of using tcdC (+117) as a specific detection site for Clostridium difficile of type 027, a highly productive strain, is also controversial. In addition, the clostridium difficile detection reagent products of the high-yield strain based on the tcdC (fatin 117) locus on the current market are too high in charge to be beneficial to wide clinical application. Thus, there is a need to further find new specific nucleic acid sequences for the detection of clostridium difficile, in particular of the high-yielding strain clostridium difficile, in order to develop specific detection cassettes for the relevant strains.
Disclosure of Invention
The invention aims to provide a novel specific sequence for high-yield clostridium difficile and to perform typing on clostridium difficile based on the specific sequence so as to realize detection of the high-yield clostridium difficile.
The invention adopts the following technical scheme:
a specific sequence of clostridium difficile with high toxigenic activity has a nucleotide sequence shown as SEQ ID No. 1.
SEQ ID No.1：TGATATAAGTGCAATAAAAAGTGCATCTCTTAGTTACTATGCTGATGAATCCAAGTATACTGATGGAGGAATGATATCATGGGTAAAGAAAGATGGAAAAATAATAATAAATGGGGGTTTTAAAGATGACCCATTAGCAGAT。
Further, the clostridium difficile with high toxigenic activity is a RT027/ST1 strain or a RT078/ST11 strain.
A primer pair for real-time fluorescent quantitative PCR amplification of the specific sequence is provided, and the nucleotide sequences of the primer pair are respectively shown as SEQ ID No.2 and SEQ ID No. 3.
SEQ ID No.2：TGATATAAGTGCAATAAAAAGTGCATCTCTTAGT。
SEQ ID No.3：ATCTGCTAATGGGTCATCTTTAAAACCC。
A probe for detecting the specific sequence by real-time fluorescence quantitative PCR has a nucleotide sequence shown as SEQ ID No. 4.
SEQ ID No.4：
5`6-FAM-CAAGTATACTGATGGAGGAATGATATCATGGGTAAA -3`BHQ1。
Wherein FAM is a fluorescence reporter group and BHQ1 is a fluorescence quencher group.
The use of a specific sequence of a highly virulent clostridium difficile as described above in the identification and typing of clostridium difficile.
A method for identifying and typing a highly productive clostridium difficile comprising the steps of:
(1) PCR amplification is carried out on genomic DNA of a sample or a bacteria to be detected by using a primer pair shown as SEQ ID No.2 and SEQ ID No.3 and a probe shown as SEQ ID No. 4;
(2) Observing the amplification curve, and if a typical S-shaped amplification curve appears, and the detection result is positive, judging that the sample contains the clostridium difficile with high toxicity or judging that the to-be-detected bacterium is the clostridium difficile with high toxicity.
A detection kit comprising the above primer pair.
A detection kit comprising the above probe.
A detection kit comprising the above primer pair and a probe. The invention has the beneficial effects that: the invention can be used for accurate and rapid nucleic acid detection of high-yield clostridium difficile, and effectively improves the identification level of clostridium difficile infection.
Drawings
FIG. 1 is a graph comparing secreted proteins predicted by genes of RT027 and RT017 strains with membrane-associated proteins.
FIG. 2 shows the specific site mutation (165℃ Fwdarw.T) of the differential gene of the RT027/ST1 strain.
FIG. 3 shows the amplification results of differential genes in different strains.
FIG. 4 is a comparison of the sequencing results of differential genes in actual strains. FIG. 5 is an amplification curve of real-time fluorescent PCR detection of highly virulent 027 strain and other non-highly virulent strains using primer pairs and probes.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
1. Prediction of protein localization and differential membrane protein gene analysis
According to the invention, 4 strains of high-virulence clostridium difficile RT027/ST1 and 3 strains of common clostridium difficile RT017/ST37 are selected for whole genome sequencing analysis, and protein positioning prediction is performed.
The results show that: the secretory proteins of the high-toxigenic RT027/ST1 strain have 158 secretory proteins and 903 membrane-related proteins; the common clostridium difficile RT017/ST37 strain has 145 secreted proteins and 888 membrane-associated proteins.
By comparing the secreted proteins of the RT027/ST1 and RT017/ST37 strains with the membrane-associated proteins, respectively, it was found that 43 secreted proteins and 89 membrane-associated proteins were specifically present in the RT027/ST1 bacteria; there were 30 secreted proteins and 74 membrane associated proteins specifically present in the RT017/ST37 strain (as shown in FIG. 1).
Among the membrane-associated proteins, we found that the gene (Genebank: VFC 12735.1) was specifically present in the RT027/ST1 type bacteria, whereas it was absent in the RT017/ST37 strain.
2. Sequence characterization of the Gene (Genebank: VFC 12735.1) on other strains was analyzed by alignment (BLAST) using an on-line database (NCBI)
To verify whether this gene is specific in the RT027/ST1 and RT017/ST37 strains, we used this gene sequence for BLAST sequence alignment in NCBI database. The 83 genes (Identity score > 85) with higher consistency with the search sequence obtained by the selective alignment were analyzed. It was found that 24 strains among 25 RT027/ST1 strains had a specific site mutation (165C > T) in the gene. In other strains, only the RT078/ST11 strain had this mutation (see FIG. 2).
In view of the fact that both RT078/ST11 and RT027/ST1 strains are highly virulent pathogenic bacteria responsible for severe CDI infection, this mutation site is believed to be of great significance for the detection and identification of Clostridium difficile highly virulent strains.
3. Actual confirmation of sequence specificity of the Gene Using the actual Strain
To further verify whether this gene has specific presence or absence in the actual strains of RT027/ST1, RT017/ST37, etc., we designed PCR primers:
the pre-primer: ACTCTAGTGGAATTATTAGTAGTAATTGCTATAA;
and (3) a rear primer: ATCTAGACCACTTTTGCTTATTTCTCCTT.
The above primers were used to carry out ordinary PCR amplification of the locally isolated strains of ST1, ST2, ST3, ST37, ST38 and ST 54.
The total reaction system is25. Mu.L, including 12.5. Mu.L of 2 XTaq Master Mix, 1. Mu.L of each of the upstream and downstream primers (10 pmol/L), ddH 2 O8.5. Mu.L, template DNA 2. Mu.L. The reaction conditions are as follows: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 50s, for a total of 30 cycles; finally, the extension is carried out at 72 ℃ for 10min. The resulting PCR product was electrophoresed on a 1% agarose gel and imaged. And after the quality is qualified, sequencing and sequence comparison are carried out on the amplified products.
As a result, it was found that the gene was indeed deleted in other RT017/ST37 strains isolated locally (FIG. 3). Furthermore, the gene locus mutation (165C > T) was also specific in the RT027/ST1 strain of the locally isolated strain (FIG. 4).
4. Designing specific fluorescent quantitative PCR primers and probes for detecting high-yield strain RT027/ST1 and RT078/ST11, and detecting and verifying actual strain
To further confirm the identification of the RT027/ST1 strain by the mutation site (165C > T) of the gene, we designed primer pairs (SEQ ID No.2 and SEQ ID No. 3) and probes (SEQ ID No. 4) for the above mutation sites, and amplified and sequenced the different species of strains (ST 1, ST2, ST3, ST37, ST38, ST 54) that were physically isolated locally.
The total reaction system was 20. Mu.L, which comprises 10. Mu.L of a 2 XTaq Master Mix solution, 1. Mu.L of each of the upstream and downstream primers and probes (10 pmol/L), ddH 2 O5. Mu.L, template DNA 2. Mu.L. The reaction conditions are as follows: pre-denaturation at 95℃for 3min; denaturation at 95℃for 5s and annealing at 65℃for 30s were performed for 40 cycles in total.
As a result, it was found that a typical S-type amplification curve appears on the amplification curve of ST1 (strain type 027), indicating that the detection result was positive. The amplification curve is shown in FIG. 5. In summary, the specific nucleotide sequence (SEQ ID No. 1) specifically identifies Clostridium difficile RT027/ST1 strain with high virulence. Based on the results of the detection of the RT027/ST1 strain and the identity of SEQ ID No.1 between the RT078/ST11 and RT027/ST1 strains, the specific nucleotide sequence (SEQ ID No. 1) also makes it possible to specifically identify the highly virulent Clostridium difficile RT078/ST1 strain.
SEQUENCE LISTING
<110> second Hospital of university of Hebei medical science, fourth Hospital of Hebei medical science
<120> specific sequences of clostridium difficile with high toxigenic activity and their use in identification and typing
<130> 2022
<160> 4
<170> PatentIn version 3.3
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<213> Synthesis
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Claims (4)
1. The primer probe group for detecting the clostridium difficile with high toxigenic activity based on the real-time fluorescence quantitative PCR is characterized by comprising primer pairs shown as SEQ ID No.2 and SEQ ID No.3 and probes shown as SEQ ID No.4, wherein the clostridium difficile with high toxigenic activity is a RT027/ST1 strain or a RT078/ST11 strain.
2. The application of a primer probe group for detecting clostridium difficile with high toxigenic activity based on real-time fluorescence quantitative PCR is characterized in that the primer probe group comprises primer pairs shown as SEQ ID No.2 and SEQ ID No.3 and probes shown as SEQ ID No.4, and if the real-time fluorescence quantitative PCR detection curve result is positive, the sample is judged to contain clostridium difficile with high toxigenic activity or the clostridium difficile to be detected is judged to be clostridium difficile with high toxigenic activity, and the clostridium difficile with high toxigenic activity is RT027/ST1 strain or RT078/ST11 strain.
3. A method for identifying and typing clostridium difficile with high virulence for non-disease diagnostic purposes, comprising the steps of: (1) Carrying out real-time fluorescent quantitative PCR amplification on genomic DNA of a sample or a bacteria to be detected by using a primer pair shown as SEQ ID No.2 and SEQ ID No.3 and a probe shown as SEQ ID No. 4; (2) If the real-time fluorescence quantitative PCR detection curve results are positive, the sample is judged to contain high-yield clostridium difficile or the to-be-detected bacterium is judged to be high-yield clostridium difficile, and the high-yield clostridium difficile is RT027/ST1 strain or RT078/ST11 strain.
4. A detection kit comprising the primer-probe set of claim 1.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012087135A1 (en) * | 2010-12-22 | 2012-06-28 | Academisch Ziekenhuis Leiden H.O.D.N. Lumc | Genetic markers specific for clostridium difficile ribotypes 027 (nap01/b1; rt 027) and 078 (nap7/8; rt 078) and their use |
| WO2014061783A1 (en) * | 2012-10-19 | 2014-04-24 | 株式会社イーベック | Human antibody specific to toxin produced from clostridium difficile, or antigen-binding fragment thereof |
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