Digital PCR joint detection method and detection kit for four virulence-related genes of clostridium difficile

Abstract

The invention discloses a digital PCR (Polymerase Chain Reaction) joint detection method for four virulence related genes (tcdA, tcdB, tcdC and cdtB) of clostridium difficile and a kit thereof. The detection system comprises a specific primer pair as shown in SEQ ID NO: 1-13 and a fluorescence labeling probe group. Through a digital PCR technology platform, multi-target and high-sensitivity absolute quantitative detection is realized, interference of inhibitors in a sample can be effectively overcome, detection is completed within 4 hours, accurate pathogen load information is provided for clinic, and rapid diagnosis and treatment evaluation are assisted.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of PCR detection technology, and particularly relates to a digital PCR combined detection method for four virulence-related genes of Clostridium difficile and a detection kit thereof. BACKGROUND

[0002] Clostridium difficile infection (CDI) is a serious healthcare-associated infection with clinical manifestations ranging from asymptomatic carriage to life-threatening pseudomembranous colitis, and is characterized by high recurrence rate, high mortality rate and high medical burden. The incidence of CDI continues to rise worldwide, and the spread of high virulence strains (such as ribosome type 027, type 002 and ST81) further exacerbates the difficulty of prevention and control.

[0003] Current diagnostic methods for CDI mainly include cytotoxicity neutralization test (limited sensitivity and complex operation), toxin culture (requires strict anaerobic environment and long cycle), GDH ELISA (poor specificity, cannot distinguish between toxin-producing strains and non-toxin-producing strains), and direct detection of toxin ELISA (low sensitivity). Molecular detection methods such as qPCR have improved sensitivity and speed, but multiple qPCR has problems such as complex primer design, product inhibition and limited quantitative accuracy. Recombinase polymerase amplification (RPA) technology is suitable for rapid screening, but only qualitative detection can be achieved and the detection target is limited.

[0004] Although the prior art has its advantages, it still has the following common deficiencies: the number of detection targets is small (usually only 1-2 genes), absolute quantification is difficult to achieve, the sensitivity for low copy samples is insufficient, and it is easily interfered by inhibitors in clinical samples. Therefore, it is of great significance to develop a new detection technology that can simultaneously detect multiple virulence genes, achieve absolute quantification, and have strong anti-interference ability. SUMMARY

[0005] The purpose of the present application is to overcome the deficiencies in the prior art and provide a digital PCR combined detection method for four virulence-related genes of Clostridium difficile and a detection kit thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present application is as follows: The first aspect is to provide a primer set for digital PCR combined detection of four virulence-related genes of Clostridium difficile, which comprises the following primer pairs: a tcdA gene upstream primer with a sequence as shown in SEQ ID NO: 1, and a tcdA gene downstream primer with a sequence as shown in SEQ ID NO: 2; a tcdB gene upstream primer having a sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5, and a tcdB gene downstream primer having a sequence as shown in SEQ ID NO: 6; a tcdC gene upstream primer having a sequence as shown in SEQ ID NO: 8, and a tcdC gene downstream primer having a sequence as shown in SEQ ID NO: 9; a cdtB gene upstream primer having a sequence as shown in SEQ ID NO: 12, and a cdtB gene downstream primer having a sequence as shown in SEQ ID NO: 13.

[0007] The second aspect is to provide an oligonucleotide probe set for digital PCR combined detection of four virulence-related genes of Clostridium difficile, the probe set comprising: a tcdA gene probe having a sequence as shown in SEQ ID NO: 3; a tcdB gene probe having a sequence as shown in SEQ ID NO: 7; a tcdC gene probe 1 having a sequence as shown in SEQ ID NO: 10, and a tcdC gene probe 2 having a sequence as shown in SEQ ID NO: 11; a cdtB gene probe having a sequence as shown in SEQ ID NO: 14, SEQ ID NO: 15; wherein the 5' end of each probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group.

[0008] Further, the fluorescent reporter group is selected from any one of FAM, ROX, HEX, TET, Cy5 or VIC; and the quencher group is selected from any one of TAMRA, BHQ1, BHQ2, BHQ3 or MGB.

[0009] The third aspect is to provide an oligonucleotide set for digital PCR combined detection of four virulence-related genes of Clostridium difficile, comprising the primer set and the oligonucleotide probe set described above.

[0010] The fourth aspect is to provide a combined detection kit for four virulence-related genes of Clostridium difficile, comprising the oligonucleotide set described above.

[0011] Further, the combined detection kit further comprises a DNA polymerase; and the DNA polymerase is one of Taq enzyme, TTh enzyme or TTX enzyme.

[0012] The fifth aspect is to provide a digital PCR combined detection method for four virulence-related genes of Clostridium difficile, comprising the following steps: Step 1, extracting genomic DNA of the sample to be tested; Step 2, using the DNA obtained in step 1 as a template, configuring a digital PCR reaction system, wherein the reaction system comprises the above-mentioned primer set and the above-mentioned oligonucleotide probe set; Step 3, performing a digital PCR amplification reaction; Step 4, after the reaction is completed, collecting the fluorescence signal, analyzing the results, and realizing quantitative detection of the tcdA, tcdB, tcdC and cdtB genes in the sample to be tested.

[0013] Compared with the prior art, the above technical scheme has the following technical effects: Based on the digital PCR technology, the present application establishes a combined detection system for four virulence-related genes (tcdA, tcdB, tcdC and cdtB) of Clostridium difficile, which has the following beneficial effects: 1) Multi-target detection, improving the amount of detection information and sensitivity: breaking through the limitation of the existing method of detecting only 1-2 targets, the simultaneous detection of four virulence genes can be realized, the amount of detection information is significantly increased, the detection sensitivity of the target strain is improved, and the clinical result interpretation and precise treatment are assisted.

[0014] 2) The advantage of the digital PCR platform is outstanding: with the limited dilution and absolute quantification capability of digital PCR, the interference of inhibitors in clinical samples can be effectively overcome, the target gene copy number can be stably output, and the pathogen load can be accurately quantified, which provides a basis for clinical treatment evaluation.

[0015] 3) Rapid detection and simple operation: the detection can be completed within 4 hours and a report can be issued, which significantly shortens the detection time compared with the traditional method and is more suitable for the clinical rapid diagnosis demand. DETAILED DESCRIPTION

[0016] The present application will be further described below in conjunction with specific examples, but not as a limitation of the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.

[0017] The experimental methods in the following examples not specified in the specific conditions are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0018] The four virulence-related genes described in the present application are tcdA, tcdB, tcdC (i.e. tcdCΔ117) and cdtB.

[0019] Example 1: Digital PCR combined detection method for four virulence-related genes (tcdA, tcdB, tcdC and cdtB) of Clostridium difficile The detection system for Clostridium difficile, combined with the Leading Gene multicolor fluorescent channel digital PCR platform, provides a combined detection method for four virulence-related genes. The configuration method and PCR amplification procedure of the detection system are as follows (Tables 1-4): Table 1. Multiplex detection system for Clostridium difficile

[0020] Table 2 Enzyme Mixture Formulation

[0021] Table 3 Primer-probe mix formulations

[0022] Table 4. Primer and probe sequences

[0023] PCR amplification program: 95℃, 10min; [98℃, 15sec, 55℃, 30sec, 72℃, 30sec]*40cycles.

[0024] In this embodiment, 50 clinical stool samples were collected for methodological comparison. Toxin culture was used as the gold standard for the diagnosis of Clostridium difficile. Compared with other traditional methods such as Clostridium difficile antigen detection (GDH), toxin detection (Toxin) and culture methods, digital PCR showed high sensitivity and specificity (Table 5), with a sensitivity of 94% and a specificity of 87.5%, which can well meet the clinical requirements.

[0025] Table 5

[0026] Example 2: Combined detection kit for four virulence-related genes (tcdA, tcdB, tcdC, and ctdB) in Clostridium difficile This kit consists of primer-probe mixture, enzyme mixture, internal control, ultrapure water, and positive control. The components are shown in Table 6 below. Table 6

[0027] Example 3: Detection method and result interpretation of the kit 1) Extraction of fecal DNA Take 25µL of watery fecal sample and 25mg of solid fecal sample for rapid fecal DNA extraction. Use the QIAGEN Fecal DNA Extraction Kit (QIAamp® Power Fecal DNA Kit, catalog number: 166014313) to extract DNA as a template for later use.

[0028] 2) Digital PCR reaction The digital PCR reaction was performed with the extracted DNA as template. The PCR amplification procedure was as follows: 95℃, 10min; [98℃, 15sec; 55℃, 30sec; 72℃, 30sec] *40 cycles, the reaction system was 3.6μL of enzyme mixture, 1.8μL of primer probe, 1μL of internal reference, 5μL of template, and water was added to 15μL.

[0029] 3) Chip reading and analysis After the completion of the digital PCR reaction, the chip reading software GeneDPT.exe was opened for chip analysis.

[0030] 4) Result interpretation The detection lower limit of virulence genes tcdA, tcdB, ctdB and tcdCΔ117 was 0.35, 0.4, 0.4 and 0.3 (copies / μL) respectively. The detection of the gene was positive when it exceeded the detection lower limit, and the specific value was reported.

[0031] The results are as follows: Toxin-producing C. difficile was not detected (tcdA-, tcdB-, ctdB- and tcdCΔ117-); Only B toxin-producing C. difficile was detected (tcdA-, tcdB+, ctdB- and tcdCΔ117-); Toxin-producing C. difficile was detected (tcdA+, tcdB+, ctdB- and tcdCΔ117-); High virulence ribosome 027 type C. difficile was detected (tcdA+, tcdB+, ctdB+ and tcdCΔ117+); Single tcdA, ctdB and tcdCΔ117 positive, re-detection was required.

[0032] The above merely describes the preferred embodiments of the present application, and does not limit the embodiments and protection scope of the present application. It should be realized by those skilled in the art that any equivalent replacement and obvious changes made according to the content of the present application should be included in the protection scope of the present application.

Claims

1. A primer set for the combined digital PCR detection of four virulence-related genes in Clostridium difficile, characterized in that, The primer set includes the following primer pairs: The upstream primer of the tcdA gene, the sequence of which is shown in SEQ ID NO:1, and the downstream primer of the tcdA gene, the sequence of which is shown in SEQ ID NO:2; The upstream primer of the tcdB gene, the sequence of which is shown in SEQ ID NO:4 and SEQ ID NO:5, and the downstream primer of the tcdB gene, the sequence of which is shown in SEQ ID NO:6; The upstream primer of the tcdC gene, the sequence of which is shown in SEQ ID NO:8, and the downstream primer of the tcdC gene, the sequence of which is shown in SEQ ID NO:9; The upstream primer of the cdtB gene has the sequence shown in SEQ ID NO:12, and the downstream primer of the cdtB gene has the sequence shown in SEQ ID NO:

13.

2. An oligonucleotide probe set for the combined digital PCR detection of four virulence-related genes in Clostridium difficile, characterized in that, The probe set includes: The tcdA gene probe, the sequence of which is shown in SEQ ID NO:3; The tcdB gene probe, the sequence of which is shown in SEQ ID NO:7; tcdC gene probe 1, the sequence of which is shown in SEQ ID NO:10, and tcdC gene probe 2, the sequence of which is shown in SEQ ID NO:11; The cdtB gene probe has the sequence shown in SEQ ID NO:14 and SEQ ID NO:

15. Each probe has a fluorescent reporter group labeled at its 5′ end and a quencher group labeled at its 3′ end.

3. The oligonucleotide probe set according to claim 2, characterized in that, The fluorescent reporter group is selected from any one of FAM, ROX, HEX, TET, Cy5 or VIC; the quencher group is selected from any one of TAMRA, BHQ1, BHQ2, BHQ3 or MGB.

4. An oligonucleotide genome for the combined digital PCR detection of four virulence-related genes in Clostridium difficile, characterized in that, It comprises the primer set as described in claim 1 and the oligonucleotide probe set as described in claim 2 or 3.

5. A combined detection kit for four virulence-related genes in Clostridium difficile, characterized in that, It comprises the oligonucleotide group as described in claim 4.

6. The combined detection kit according to claim 5, characterized in that, It also contains a DNA polymerase; the DNA polymerase is one of Taq polymerase, TTh polymerase or TTX polymerase.

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