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Parallel probe, gene chip, kit and assay method for clostridium difficile assay

A technology for detecting gene chips and detection kits, applied in the field of molecular biology, can solve the problems of insufficient sensitivity, short detection period, low specificity, etc., and achieve the effects of improving accuracy and precision, rapid detection, and high specificity

Pending Publication Date: 2017-06-20
QINGDAO JIAOZHOU CENT HOSPITAL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The currently commonly used rapid detection methods for Clostridium difficile mainly include the following: 1) Cytotoxicity test (CCTA): Cytotoxicity test is to detect cell lesions caused by toxins A and B, but it requires corresponding facilities for cell culture and microscope observation 2) Enzyme-linked immunoassay (EIA) detection of toxin A / B: this method has high specificity, can distinguish toxin-producing strains from non-toxin-producing strains, and the detection cycle is short, and can be detected within a few hours As a result, Meridian Premier, TechLab Tox A / B Quik Chek, TeehLab ToxA / B II and other commercial kits have been put into use, but the disadvantage of this method is that the sensitivity is not enough, and strains cannot be obtained for molecular typing, etc. Further research; 3) Enzyme-linked immunoassay (EIA) detection of glutamate dehydrogenase (GDH): GDH is an antigenic protein present in the cell wall of Clostridium difficile, which is more sensitive than EIA for detecting toxin A / B, but the The specificity of the method is low; 4) PCR detection: real-time PCR detection technology has the characteristics of high sensitivity and high specificity, and the method can get the result within 3 hours, and the method is simple to use

Method used

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  • Parallel probe, gene chip, kit and assay method for clostridium difficile assay
  • Parallel probe, gene chip, kit and assay method for clostridium difficile assay
  • Parallel probe, gene chip, kit and assay method for clostridium difficile assay

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 Design of primers and probes for target fragments

[0023] Find the gene sequence of Clostridium difficile in the NCBI database, select the highly specific gene sequence as the target sequence (ID number: NC_009089.1), and design primers and probes. After the target sequence is determined, according to the primer and probe design principles, design the amplification primers and probes of the C. difficile target gene. The 5'end of the R primer of the C. difficile target gene amplification primer is labeled with Cy3. The specific sequence is as follows :

[0024] C-DIFF target sequence amplification primers:

[0025] F primer (C-DIFF-F): 5'-gagagtttgatyctggctcag-3' (SEQ ID NO. 8);

[0026] R primer (C-DIFF-R): 5'-Cy3-aaggaggtgatccarccgca-3' (SEQ ID NO. 9).

[0027] C-DIFF parallel probe:

[0028] C-DIFF probe 1:

[0029] 5’-ttttttttttttttttcagcaacgccgcgtgagtgatgaaggccttcgggtcgtaa-3’ (SEQ IDNO.1)

[0030] C-DIFF probe 2:

[0031] 5’-ttttttttttttttttaccagttgcgaaggcggctctctgga...

Embodiment 2

[0042] Example 2 Preparation of chip

[0043] Spot the parallel probes of C-DIFF in Example 1 on the amino-modified glass substrate in accordance with the order in Table 1, to obtain a gene chip containing probes (such as figure 1 Shown). The probe concentration is 30uM, each spot is 0.2uL, and then incubated at 80°C for 1.5 hours.

[0044] Table 1 The sequence of the probes of the Clostridium difficile detection chip

[0045]

Embodiment 3

[0046] Example 3 Detection method

[0047] 1. Extract the genome of the sample to be tested

[0048] Use the viral genomic DNA / RNA co-extraction kit to extract the genome of the sample to be tested as the amplification template according to the steps in the operating instructions.

[0049] 2. PCR amplification of the target fragment

[0050] After a lot of experiments, the PCR amplification system and PCR reaction program of the target gene detected by C-DIFF were determined. The PCR amplification system with a total volume of 10 μL contains the following reagents:

[0051]

[0052] The PCR reaction program is: 95°C 5min; 95°C 10s, 55°C 30s, 72°C 30s, 30 cycles; 72°C 10min.

[0053] 3. Hybridization of PCR product and chip

[0054] Follow the steps below: a. On the gene chip prepared in Example 2, spot the PCR amplification products of the sample to be tested into the detection holes of 5 probes, and at the same time, the positive control sample and the blank control sample The PCR prod...

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Abstract

The invention discloses a parallel probe for clostridium difficile assay. The parallel probe for clostridium difficile assay consists of a probe 1, a probe 2, a probe 3, a probe 4 and a probe 5, the sequences of which are SEQ ID NO.1-5. The invention further discloses a gene chip containing the parallel probe and a kit for the gene chip. The invention further discloses a method for assaying clostridium difficile by utilizing the kit, which includes the following steps: the clostridium difficile assay chip disclosed by the invention is used, and when results displayed by three or more of the five assay probes are positive, a judgment is made that a sample is infected with clostridium difficile; when assay results displayed by less than three of the five assay probes are positive, whether the sample is infected with clostridium difficile cannot be judged, and the assay needs to be repeated or another assay method needs to be used for further verification. The invention is easy to operate, and is highly efficient and highly accurate, and results are easy to read.

Description

Technical field [0001] The invention belongs to the field of molecular biology and relates to a parallel probe, gene chip, kit and detection method for detecting Clostridium difficile. Background technique [0002] Clostridium difficile (clostridium difficile) is an obligate anaerobic gram-positive Clostridium spores, generally considered to be the normal flora in the environment and human intestines. Long-term application of antibiotics, immunosuppressants or chemotherapeutic drugs to cause the drug-resistant Clostridium difficile toxin-producing strains to overproduce and release toxins is the main factor leading to Clostridium difficile-related diarrhea (CDAD). Clostridium difficile is mainly caused by the production of A, B and binary toxin (CDT). Toxin A, also known as enterotoxin, causes neutrophil infiltration in the intestinal wall of the ileum, releasing lymphokines, causing massive secretion of fluid and hemorrhagic necrosis; toxin B, also known as cytotoxin, can depol...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C40B40/06C12N15/11
CPCC12Q1/689C12Q1/686C40B40/06
Inventor 李修敏申友亮朱同娥
Owner QINGDAO JIAOZHOU CENT HOSPITAL
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