PCR-internal control nucleic acid test strip detection method for mycoplasma pneumoniae detection

A technology of mycoplasma pneumoniae and nucleic acid test strips, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, and microorganism measurement/inspection, and can solve problems such as fluorescent probes, expensive instruments, and complicated operations

Inactive Publication Date: 2019-05-14
XIAN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Fluorescent quantitative PCR is the most commonly used method in MP-DNA detection, but the method is complicated to operate, and there are problems such as fluorescent probes and expensive instruments, and many grassroots clinical units are still unable to carry out

Method used

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  • PCR-internal control nucleic acid test strip detection method for mycoplasma pneumoniae detection
  • PCR-internal control nucleic acid test strip detection method for mycoplasma pneumoniae detection
  • PCR-internal control nucleic acid test strip detection method for mycoplasma pneumoniae detection

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Experimental program
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Effect test

Embodiment 1

[0067] 1. The scope of samples is applicable to sputum samples from patients suspected of Mycoplasma pneumoniae infection. Depending on the difficulty of dissolving the sputum specimen, add trypsin of 1 to 3 times the volume of the sputum, and remove the sputum after centrifugation at 2000-4000rpm for 2min, then take 1ml of the supernatant and centrifuge at 10000~15000rpm for 5min, discard the supernatant and add it to the pellet 500μl DNA lysis buffer (0.5% NP-40 solution with proteinase K concentration of 200ng / ml) shake and mix well, put in a 56℃ water bath for 1h, boil for 10min, centrifuge at 10000-13000rpm for 5min, take the supernatant as template solution for amplification detection .

[0068] 2. Take the PCR centrifuge tube and add the reagents according to the following table

[0069]

[0070] 3. Put the PCR tube into the PCR amplification instrument and perform amplification according to the following procedures:

[0071] UNG enzyme 50℃2min; 95℃pre-denaturation 5min; 94℃...

Embodiment 2

[0074] 1. The sample scope is applicable to throat swab samples from patients suspected of Mycoplasma pneumoniae infection. Use 1ml saline to soak the throat swab cotton swab, after repeated squeezing, discard the cotton swab, shake for 30-60s, centrifuge at 10000~15000rpm for 5min, discard the supernatant and add 50-200μl DNA lysis solution to the pellet (proteinase K concentration is 200ng / ml 0.5% NP-40 solution), boil for 10 min, centrifuge at 10000-13000 rpm for 5 min, and take the supernatant as a template solution for amplification detection.

[0075] 2. Take the PCR centrifuge tube and add the reagents according to the following table

[0076]

[0077] 3. Put the PCR tube into the PCR amplification instrument and perform amplification according to the following procedures:

[0078] UNG enzyme 50℃2min; 95℃pre-denaturation 5min; 94℃denaturation 30s, 54℃annealing 30s, 72℃ extension 1min, for 30 cycles; 72℃ full extension 1min.

[0079] 4 Drop the amplified product of the PCR tub...

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Abstract

The invention discloses a mycoplasma pneumoniae detection method based on a PCR-internal control nucleic acid test strip. The method specifically comprises the following steps that an MP specific primer modified with digoxin and biotin is designed according to the 16S rRNA nucleic acid sequence of mycoplasma pneumoniae, and a beta globin gene primer modified with digoxin and fluorescein is designed according to the nucleic acid sequence of a human beta globin gene; the DNA of a to-be-detected sample is extracted and serves as a template, the MP specific primer and the beta globin gene primer are used for PCR amplification to obtain an amplified product of the DNA of the to-be-detected sample, and the UNG enzyme anti-pollution technology and internal control are added for avoiding false positive and false negative results respectively. The amplified product of the DNA of the to-be-detected sample is detected through the mycoplasma pneumoniae detection internal control nucleic acid teststrip. The mycoplasma pneumoniae detection method based on the PCR-internal control nucleic acid test strip is easy to operate and capable of quickly detecting mycoplasma pneumoniae.

Description

Technical field [0001] The invention belongs to the technical field of molecular biology detection, and relates to a method for detecting Mycoplasma pneumoniae based on PCR-internal control nucleic acid test strips. Background technique [0002] Mycoplasma pneumoniae (MP) is one of the most common pathogenic microorganisms that cause respiratory tract infections. Its current incidence is showing an upward trend and can cause multiple system diseases. Mycoplasma pneumoniae lacks cell walls, making it intrinsically resistant to antibiotics that affect cell wall synthesis. Clinically, drugs that inhibit or affect protein and nucleic acid synthesis should be selected for treatment, such as macrocyclic vinegars, phenosterones, and aminoglycosine. Class, tetracyclines, etc. Since most drugs have adverse effects on the normal growth and development of children, macrolides are currently the first choice. With the widespread use of macrocyclic antibiotics, the resistance of Mycoplasma p...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/35
Inventor 廉婷陈程姚杨邱忠营李慧瑾李银未
Owner XIAN MEDICAL UNIV
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