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Multiplex polymerase chain reaction (mPCR)-denaturing high-performance liquid chromatography (DHPLC) primers and method for detecting and identifying mycobacterium

A technology of mycobacterium and mycobacterium avium, which is applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of lack of standardization and impact of standardization of detection methods and kits, and achieve saving Reagent cost, reduced workload, good specificity

Inactive Publication Date: 2013-11-20
INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the development of molecular biology technology, many laboratories at home and abroad have carried out research on PCR methods for tuberculosis and paratuberculosis, but the lack of standardization of detection methods and kits is the key to affecting the accuracy of PCR results

Method used

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  • Multiplex polymerase chain reaction (mPCR)-denaturing high-performance liquid chromatography (DHPLC) primers and method for detecting and identifying mycobacterium
  • Multiplex polymerase chain reaction (mPCR)-denaturing high-performance liquid chromatography (DHPLC) primers and method for detecting and identifying mycobacterium
  • Multiplex polymerase chain reaction (mPCR)-denaturing high-performance liquid chromatography (DHPLC) primers and method for detecting and identifying mycobacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Preparation of primers for mycobacteria and pathogenic mycobacteria mPCR-DHPLC detection method

[0067] According to the sequence of Mycobacterium-specific 16s rRNA, one of the sequences (GeneBank No.CP000611) was selected, and the sequence was designed as shown in SEQ ID No.1 and SEQ ID No.2 according to the annealing temperature of the primer at about 60°C. The primer pair shown, the PCR product length is 230bp:

[0068] Upstream primer (Primer 1) SEQ ID No.1: CGTGCGGGCGATAC;

[0069] Downstream primer (Primer 2) SEQ ID No. 2: GGCACGGATCCCAA.

[0070] According to the specific sequences IS6110 and IS1081 of the Mycobacterium tuberculosis complex, one of the sequences (CP003248.1) was selected, and the sequences of IS6110 and IS1081 were designed according to the annealing temperature of the primers at about 60°C, such as SEQ ID No.4 and For the primer pairs shown in SEQ ID No.5, SEQ ID No.7 and SEQ ID No.8, the PCR product lengths are 178bp and 379bp res...

Embodiment 2

[0082] Example 2: Establishment and optimization of mycobacterial five-fold mPCR-DHPLC method PCR detection reaction system

[0083] 1. Method

[0084] 1. Optimization of primer concentration

[0085] In the experiment, the primer concentration was increased from 0.1 μmol / L to 0.6 μmol / L by 0.1 μmol / L. The matrix method was used to conduct the comparison experiment, and the other conditions of the comparison experiment were completely consistent.

[0086] 2. Mg 2+ Concentration optimization

[0087] Mg 2+ It will affect the specificity and amplification efficiency of the PCR reaction. In the case of fixing other reaction components unchanged, Mg 2+ Concentration gradient, for Mg 2+ concentration was optimized, Mg 2+ The concentration starts from 1.5mmol / L and increases by 0.5mmol / L to 6mmol / L.

[0088] 3. Optimization of PCR reaction conditions

[0089] In order to improve the sensitivity and specificity of the PCR reaction, according to the designed primer annealing t...

Embodiment 3

[0100] Embodiment 3: The inventive method detects clinical sample

[0101] 1. Sample collection

[0102] Sputum: There is very little phlegm in cows, so it is advisable to collect the sputum in the early morning. Use a rubber tube to extend from the mouth to the trachea, and connect the outer end to a syringe to suck the sputum. Sputum lumps coughed up by cows can also be taken; human sputum samples are collected according to routine hospital operations.

[0103] 2. Sample processing

[0104] Treatment of sputum:

[0105] (1) Add 2 to 3 times the sample volume of 4g / L NaOH solution in the sample, shake well, and liquefy at room temperature for 20 minutes to make it fully liquefied; no obvious solid matter and no dragging phenomenon when sucking out means that the liquefaction is complete ; If the liquefaction is not complete, add a small amount of 4g / L NaOH solution until the liquefaction is complete.

[0106] (2) Take 1.5mL sample, centrifuge at 10,000rpm for 10min, and d...

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Abstract

The invention provides a group of nucleic acids used in a quintuple multiplex polymerase chain reaction (mPCR)-denaturing high-performance liquid chromatography (DHPLC) method for detecting mycobacterium and identifying pathogenic mycobacterium. The nucleic acids comprise five pairs of primers of which the nucleic acid sequences are shown as SEQ ID No.1 and SEQ ID No.2, SEQ ID No.4 and SEQ ID No.5, SEQ ID No.7 and SEQ ID No.8, SEQ ID No.10 and SEQ ID No.11, and SEQ ID No.13 and SEQ ID No.14, and PCR amplification products which are used as positive control and of which the nucleic acid sequences are shown as SEQ ID No.3, SEQ ID No.5, SEQ ID No.9, SEQ ID No.12 and SEQ ID No.15. The invention also provides a kit using the nucleic acids and a detection method; the method is high in specificity and flexibility and easy to operate, and high flux can be achieved; and the method has important practical significance to clinical identification on the mycobacterium infection and infectious agents of the mycobacterium infection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a five-fold mPCR-DHPLC method for synchronously detecting mycobacterium infection and identifying mycobacterium tuberculosis complex, mycobacterium paratuberculosis and mycobacterium avium. Background technique [0002] Mycobacterium (Mycobacterium), in addition to Mycobacterium tuberculosis complex (including Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium africanum, Mycobacterium microti) and Mycobacterium leprae, collectively referred to as non-tuberculous mycobacteria bacilli. According to current knowledge, there are more than 200 kinds of pathogenic and non-pathogenic mycobacteria in nature. [0003] Tuberculosis is a major infectious disease that threatens human and animal health. The World Health Organization (WHO) specially released the Global Tuberculosis Control Strategy, and designated March 24 every year as World Tuberculosis Day. Mycobacter...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 陈茹高小博马旭陆彩玲刘志玲马静云
Owner INSPECTION & QUARANTINE TECH CENT OF GUANGDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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