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Multi-fluorescent PCR kit and method for detecting clostridium difficile genes and toxin genes

A technology of multiple fluorescence and Clostridium difficile, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problem of unsatisfactory accuracy and sensitivity of CD detection, accuracy, stability and sensitivity Low, limited application and other issues to achieve the effect of avoiding false positive results, avoiding PCR false negatives, and short reporting time

Active Publication Date: 2019-08-16
GUANGZHOU SAGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing fluorescent PCR technology still has false negative or false positive, low accuracy, stability and sensitivity, which limits its clinical application.
In addition, when the sample to be tested contains complex components (such as whole blood, Imodium, Xieting, metronidazole or vancomycin, etc.), the accuracy and sensitivity of existing kits for CD detection are often not ideal

Method used

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  • Multi-fluorescent PCR kit and method for detecting clostridium difficile genes and toxin genes
  • Multi-fluorescent PCR kit and method for detecting clostridium difficile genes and toxin genes
  • Multi-fluorescent PCR kit and method for detecting clostridium difficile genes and toxin genes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] A multiplex fluorescent PCR kit for detecting Clostridium difficile genes and toxin genes, including a set of multiplex fluorescent PCR primers and probes for detecting Clostridium difficile genes and toxin genes, nucleic acid extraction reagents, and multiplex fluorescent PCR reactions Required reagents, internal standard working solution, positive control solution and negative control solution.

[0049] Wherein, the multiple fluorescent PCR primer sets and probes for detecting Clostridium difficile gene and toxin gene include tpi amplification primer pair tpi-F and tpi-R, tpi fluorescent probe tpi-P, tcdB amplification primer For tcdB-F and tcdB-R, tcdB fluorescent probe tcdB-P, internal standard primer pair HP-F and HP-R, internal standard fluorescent probe HP-P; the nucleotide sequences are respectively as SEQID NO:1 ~shown in SEQ ID NO:9.

[0050] The fluorescent substances bound to the 5' ends of the probes tpi-P, tcdB-P, and HP-P are FAM, HEX, and Cy5 respective...

Embodiment 2

[0073] The detection of the clinical sample by the kit of embodiment 2

[0074] Using the kit of Example 1 to detect Clostridium difficile in clinical samples, the specific steps are as follows:

[0075] 1. Sample settings

[0076]When collecting human feces samples, the samples should not be mixed with urine, urinal water, or disinfectants, and should not touch urinals, toilet paper, diapers, etc., to prevent the samples from being contaminated or damaged. Set 10 Clostridium difficile positive clinical samples (P1-P10) and 10 Clostridium difficile negative clinical samples (N1-N10), wherein positive samples include Clostridium difficile toxin-producing strains (P1-P5) and non-toxin-producing strains ( P6-P10). All clinical stool samples were isolated and cultured, identified by mass spectrometry, and verified by Sanger sequencing of toxins in the Laboratory Department of Guangzhou Military General Hospital.

[0077] 2. Extraction of Fecal Genomic DNA

[0078] (1) Pick 50-...

Embodiment 3

[0113] Embodiment 3 kit performance analysis

[0114] 1. Specificity analysis

[0115] (1) Cross reaction

[0116] Using the kit described in Example 1 and the detection method described in Example 2, select microorganisms that are similar to Clostridium difficile or cause similar symptoms, such as Yersinia enterocolitica, enterotoxic Escherichia coli and cereus 21 species such as Bacillus were samples to be tested, and the coincidence rate of the kit in Example 1 for detecting other pathogens similar to Clostridium difficile and causing similar symptoms was evaluated to analyze the specificity of the kit. The concentration of the sample to be tested is estimated after measuring the OD value, which is higher than 1×10 6 bacteria / mL, the results are shown in Table 7 and Figure 5 shown.

[0117] Experimental results: In addition to the amplification of the internal standard, no amplification peaks were detected for the tpi and tcdB targets, indicating that none of these sam...

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Abstract

The invention belongs to the technical field of bacterial detection, and specifically discloses a multi-fluorescent PCR kit and method for detecting clostridium difficile genes and toxin genes, wherein the kit and method not only can identify clostridium difficile but also can detect toxin B genes. The kit mainly comprises a specific primer group and a probe, the primer group and the probe are composed of a primer pair and probe for detecting clostridium difficile, a primer pair and probe for detecting toxin B, and an internal primer pair and probe. By design of the specific primers and probes, the clostridium difficile genes and the toxin genes can be detected from samples with complex components. The kit has the advantages of simple operation, short reporting time, high specificity, highsensitivity and good accuracy; PCR false negativity is avoided by introducing internal standard products, false positivity caused by contamination of amplified products is reduced, and the kit is suitable for pathogenesis screening of patients with diarrhea of unknown clinical cause.

Description

technical field [0001] The invention relates to the technical field of bacterial detection, in particular to a multiplex fluorescent PCR kit and method for detecting Clostridium difficile genes and toxin genes. Background technique [0002] Clostridium difficile (Clostridium difficile, CD), also known as Clostridium difficile, was first isolated from the feces of healthy newborns by Hall et al. in 1935. It has high nutritional requirements and is difficult to grow by conventional anaerobic culture methods, so it is difficult to isolate and cultivate , hence the name. CD is an integral part of the inherent flora in healthy humans, accounting for less than 3%. It is an opportunistic pathogenic bacteria. Under normal circumstances, microorganisms in the human intestinal tract depend on each other and restrict each other, maintaining a certain amount and proportion of micro-ecological balance. The normal intestinal flora has a certain ability to resist pathogens, which can ant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12N15/11
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/101C12Q2545/113C12Q2563/107C12N15/11C12Q1/68C12Q1/04
Inventor 罗宝花曾宏彬陈杰
Owner GUANGZHOU SAGENE BIOTECH
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