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Mycoplasma pneumoniae detection method based on PCR (polymerase chain reaction)-gold-magnetic nano immunochromatography assay

A technology of Mycoplasma pneumoniae and gold magnetic nanometers, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms, which can solve the problems of complicated operation, fluorescent probes, and expensive instruments.

Pending Publication Date: 2019-06-21
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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  • Mycoplasma pneumoniae detection method based on PCR (polymerase chain reaction)-gold-magnetic nano immunochromatography assay
  • Mycoplasma pneumoniae detection method based on PCR (polymerase chain reaction)-gold-magnetic nano immunochromatography assay
  • Mycoplasma pneumoniae detection method based on PCR (polymerase chain reaction)-gold-magnetic nano immunochromatography assay

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 1. The sample range is applicable to sputum samples from patients suspected of Mycoplasma pneumoniae infection. Use an appropriate amount of 10:1 TE buffer (PH 8.0) to liquefy the sputum, shake for 30S, centrifuge at 10000rpm for 10min, discard the supernatant, extract template DNA with a DNA extraction kit or add 20-40μL of triple distilled water to boil for 15min, 10000-13000rpm Centrifuge for 5 min, and take the supernatant as the template solution for amplification detection.

[0056] 2. Take the PCR centrifuge tube and add the reagents according to the following table

[0057]

[0058] 3. Put the PCR tube into the PCR amplifier and amplify according to the following procedures:

[0059] UNG enzyme 50℃ for 2min; pre-denaturation at 95℃ for 5min; denaturation at 94℃ for 30s, annealing at 54℃ for 30s, extension at 72℃ for 1min, 30 cycles; complete extension at 72℃ for 1min.

[0060] 4. Add the amplification product of the PCR tube dropwise to the sample pad of th...

Embodiment 2

[0062] 1. The sample range is applicable to throat swab samples from patients suspected of Mycoplasma pneumoniae infection. Use an appropriate amount of 10:1 TE buffer (PH 8.0) to soak the throat swab cotton swab, discard the cotton swab after repeated squeezing, shake for 30 s, centrifuge at 10,000 rpm for 10 minutes, extract template DNA with DNA extraction kit or add 20-40 μL of triple-distilled water and boil for 15 min , Centrifuge at 10000-13000rpm for 5min, and take the supernatant as the template solution for amplification detection.

[0063] 2. Take the PCR centrifuge tube and add the reagents according to the following table

[0064]

[0065] 3. Put the PCR tube into the PCR amplifier and amplify according to the following procedures:

[0066] UNG enzyme 50℃ for 2min; pre-denaturation at 95℃ for 5min; denaturation at 94℃ for 30s, annealing at 54℃ for 30s, extension at 72℃ for 1min, 30 cycles; complete extension at 72℃ for 1min.

[0067] 4. Add the amplification ...

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Abstract

The invention discloses a Mycoplasma pneumoniae detection method based on PCR (polymerase chain reaction)-gold-magnetic nano immunochromatography assay, which comprises the specific steps of designinga specific primer modified with digoxin and biotin according to 16S rRNA nucleotide sequence of Mycoplasma pneumoniae; extracting DNA of a sample under test, and performing PCR amplification by usingthe DNA of the sample under test as a template and the specific primer as a primer to obtain a MP target fragment amplification product of the sample under test; detecting the amplification product with a Mycoplasma pneumoniae detection gold-magnetic immunochromatographic test strip. The Mycoplasma pneumoniae detection method based on PCR-gold-magnetic nano immunochromatography assay is simple toperform and is suitable for detecting Mycoplasma pneumoniae quickly.

Description

technical field [0001] The invention belongs to the technical field of molecular biological detection, and relates to a detection method for Mycoplasma pneumoniae based on PCR-gold magnetic nano-immunochromatography. Background technique [0002] Mycoplasma pneumoniae (MP) is a common respiratory pathogen and one of the most important pathogens of community acquired pneumonia (CAP). The clinical manifestations of Mycoplasma pneumoniae infection are not characteristic, and are easily confused with respiratory tract infections caused by other viruses and bacteria, and are often mixed with other pathogenic bacteria. Therefore, a sensitive, rapid, and specific detection method is needed for early diagnosis of pneumonia. Mycoplasma pneumonia, in order to timely and correct medication, reduce and prevent drug resistance. [0003] At present, the detection methods of Mycoplasma pneumoniae mainly include isolation and culture, serological antibody detection and PCR diagnosis. Howe...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12Q1/6804C12R1/35
Inventor 廉婷余琦王荣张旭东姚杨倪菁
Owner XIAN MEDICAL UNIV
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