Nucleotides with specificity to internal transcribed spacer(ITS) of neisseria meningitidis and application thereof

A technology for Neisseria meningitidis and meningitis, which is applied to the ITS-specific nucleotides of Neisseria meningitidis and its application field, which can solve the problem of missed detection of Neisseria meningitidis and affect the reliability and sensitivity of results. It is not high enough to achieve the effect of wide market application prospect, short detection cycle and high detection accuracy

Active Publication Date: 2011-08-17
TIANJIN BIOCHIP TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It takes at least 2 to 3 days for the diagnosis of Neisseria meningitidis infection to be confirmed by this method. Serological identification is sometimes difficult to make a reliable diagnosis because of its insufficient sensitivity and many influencing factors, especially in large-scale epidemiological investigations, often due to Subjective factors such as heavy workload, inexperienced testers, and emotional fluctuations affect the credibility of the results
Traditional methods have poor specificity, low sensitivity and time-consuming
Neisseria meningitidis is very sensitive to dryness, cold, heat, sunlight, etc., and will die within 3 hours at room temperature. At the same time, it is affected by the use of antibiotics and other non-specific factors. Bacterial culture methods alone will lead to leakage of Neisseria meningitidis. check

Method used

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  • Nucleotides with specificity to internal transcribed spacer(ITS) of neisseria meningitidis and application thereof
  • Nucleotides with specificity to internal transcribed spacer(ITS) of neisseria meningitidis and application thereof
  • Nucleotides with specificity to internal transcribed spacer(ITS) of neisseria meningitidis and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 : Genome Extraction

[0059] Use a blood plate or chocolate plate to culture Neisseria meningitidis overnight at 37°C and collect the bacteria. The cells were resuspended with 500ul 50mM Tris-HCl (pH8.0) and 10ul 0.4M EDTA, incubated at 37°C for 20 minutes, and then 10ul 10mg / ml lysozyme was added to continue the incubation for 20 minutes. Then add 3ul 20mg / ml proteinase K, 15ul 10% SDS, incubate at 50°C for 2 hours, then add 3ul 10mg / ml RNase, and incubate at 65°C for 30 minutes. Add an equal volume of phenol to extract the mixture, take the supernatant, and then extract twice with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), take the supernatant, and then extract with an equal volume of ether. Extract to remove residual phenol. The supernatant was used to precipitate DNA with 2 times volume of ethanol, and the DNA was rolled out with glass wool and washed with 70% ethanol, and finally the DNA was resuspended in 30ul TE. Genomic DNA wa...

Embodiment 2

[0060] Example 2 : Amplification of ITS in Neisseria meningitidis by PCR

[0061] The ITS of Neisseria meningitidis was amplified by PCR using the genome of Neisseria meningitidis as a template. First, design an upstream primer (5'-TGT ACA CAC CGC CCG TC-3', see sequence table 1) based on the conserved region of the 16S rRNA gene at one end of the ITS, and then design a downstream primer (5'-GGT ACT TAG ATG TTT CAG TTC-3' See Sequence Listing 1). The PCR reaction program is as follows: pre-denaturation at 94°C for 5 minutes; then denaturation at 94°C for 30 seconds, annealing at 50°C for 30 seconds, and extension at 72°C for 1 minute, thus performing 30 cycles; finally, extending at 72°C for 5 minutes to obtain PCR The size and specificity of PCR products were detected by 0.8% agarose gel electrophoresis. Combine 3 tubes of PCR products, and recover and purify the PCR product bands with the UNIQ-10 Column DNA Gel Recovery Kit from Shanghai Sangon Bioengineering Technology ...

Embodiment 3

[0062] Example 3 : build its clone

[0063] The first is the acquisition of the connection product:

[0064] The PCR purified product was mixed with Promega's 3×10 -3 The pGEM-T-Easy vector was ligated at 16°C for 24 hours, with a total volume of 10 μl, which contained 1 μl of 10×buffer and 0.5 units of T4 DNA ligase to obtain the ligation product.

[0065] Followed by the preparation of competent cells:

[0066] Escherichia coli DH5α competent cells were prepared according to the method provided by Bio-Rad. Take a single colony of Escherichia coli DH5α in 5ml of LB medium, culture it at 180rpm for 10 hours, take 2ml of the culture and transfer it to 200ml of LB medium, shake vigorously at 37°C and 250rpm to about OD6000.5, then freeze The bath was cooled for 20 minutes and centrifuged at 4000 rpm for 15 minutes at 4°C. Drain the supernatant, blow off the bacteria with 200 ml of cold ice-precooled deionized sterilized water, and centrifuge at 4000 rpm for 15 minutes at 4...

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Abstract

The invention relates to nucleotides SEQ ID NO: 1 to SEQ ID NO: 3 with specificity to the 16S rRNA-23S rRNA internal transcribed spacer (ITS) of neisseria meningitidis and application thereof, and provides a fluorescent quantitative PCR(polymerase chain reaction) detection kit and a detection method by using oligonucleotide as a primer and a probe. The fluorescent quantitative PCR detection kit has the advantages of simple operation, quick result, good specificity and high sensitivity when used for detecting the neisseria meningitidis in a human body and environment, can be used in fields such as supervision and detection of foods and clinical samples, detection of pathogenic bacteria of foods, bacteriological classification and epidemiological study and has significant social benefit andgreat economic benefit.

Description

technical field [0001] The invention relates to an oligonucleotide specific to the 16S rRNA-23S rRNA intergenic region (Internal transcribed spacer, hereinafter referred to as ITS) in Neisseria meningitidis and its application. Background technique [0002] Humans are the only host of N. meningitidis. As an opportunistic pathogenic bacteria, Neisseria meningitidis often exists in the nasopharyngeal mucosa of patients or carriers. About 5-10% of healthy people carry this bacteria in the nasopharynx, and as high as 20-70% during the epidemic period , and its detoxification time can reach several weeks to 2 years. The pathogenic bacteria are directly transmitted by the air through droplets, and most infected persons have no obvious clinical symptoms, so many infected persons become asymptomatic carriers. In the non-epidemic period, the incidence rate is about 1 / 10 to 3 / 100,000, while in the epidemic period, the incidence rate can be as high as 500 / 100,000, which is extremely ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12R1/36
Inventor 王磊曹勃阳孙亚民何欣王敏
Owner TIANJIN BIOCHIP TECH CO LTD
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