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Loop-mediated isotherm amplification (LAMP) kit for detecting mycoplasma pneumoniae (Mp)

A technology of mycoplasma pneumoniae and a detection kit, which is applied in the biological field, can solve problems that have not yet been applied, and achieve the effects of easy large-scale promotion and application, increasing the possibility of pollution, and shortening the detection time

Active Publication Date: 2013-08-14
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no application of the LAMP method based on color judgment in the detection of Mycoplasma pneumoniae

Method used

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  • Loop-mediated isotherm amplification (LAMP) kit for detecting mycoplasma pneumoniae (Mp)
  • Loop-mediated isotherm amplification (LAMP) kit for detecting mycoplasma pneumoniae (Mp)
  • Loop-mediated isotherm amplification (LAMP) kit for detecting mycoplasma pneumoniae (Mp)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The design of embodiment 1 primer

[0035] In the present invention, 60 clinical strains of Mycoplasma pneumoniae are firstly isolated in China, and the full-length p1 genes of the 60 strains of Mycoplasma pneumoniae are amplified and sequenced. By comparing the p1 gene sequences of 60 domestic strains of Mycoplasma pneumoniae with the reported p1 gene sequences in the NCBI database, the most conserved region of the p1 gene was selected and specific primers were designed using Primer Explorer V3 software. The specific target sequence of the conserved region is the nucleotide sequence shown in SEQ ID No.5.

[0036] Design 4 primers, including two inner primers (FIP and BIP) and two outer primers (F3 and B3), the nucleic acid sequences of which are as follows:

[0037] F3: 5'-TCTTACCACTGTTAACGGCC-3';

[0038] B3: 5'-CCGCTTTGGTCAACACATCA-3';

[0039] FIP: 5'-ACGGCAACACGTAATCAGGTCATCCAGTCAAGGTCCCCAA-3';

[0040] BIP: 5'-AGGACTTGCCATTGGAATCCCAGATAGCGCAAACCCAGCC-3'.

Embodiment 2

[0041] Example 2 Establishment of Mycoplasma pneumoniae LAMP detection method

[0042] By setting different concentrations of MgSO 4(150mM): 0μl, 0.5μl, 1μl, 1.5μl, 2μl; different concentrations of betaine (4M): 0μl, 1μl, 2μl, 4μl, 8μl, 16μl; and 4 primer concentrations obtained in Example 1, FIP / BIP primer (100uM): 0.2μl, 0.4μl, 0.8μl, 1.6μl; F3 / B3 primer (10μM): 0.5μl, 1.0μl, 1.5μl, 2.0μl. Other conditions Select the preferred experimental conditions for the experimental procedure.

[0043] Respectively react at different temperatures (61°C to 65°C) for 1 hour, 85°C for 3 minutes, and stop the reaction. Since the amplification is fastest under the condition of 65°C for 1 hour, it is preferable to perform the reaction under this condition.

[0044] Reaction at 63°C for 1h, 85°C for 3min, for different concentrations of MgSO 4 , Betaine, and the reaction system of the concentration ratio of internal and external primers were optimized, and finally by containing 10 3 The t...

Embodiment 3

[0056] Example 3 Evaluation of the characteristics of the LAMP detection kit

[0057] 1. Sensitivity evaluation

[0058] Sensitivity (sensitivity), also known as true positive rate (true positive rate), is actually the percentage of Mycoplasma pneumoniae that is correctly judged as Mycoplasma pneumoniae according to the standard of the detection method. Nine ATCC standard strains (ATCC39505, ATCC15531, ATCC29085, ATCC29342, ATCC29343, ATCC15377, ATCC15492, ATCC49894, ATCC15293) and 50 laboratory-preserved clinical isolates of Mycoplasma pneumoniae were detected by the optimized LAMP system based on color determination. Except for the negative control, all 59 strains of Mycoplasma pneumoniae were detected positive based on the color-based LAMP system, with a sensitivity of 100%.

[0059] 2. Specificity evaluation

[0060] Specificity (specificity), also known as true negative rate (true negative rate), that is, the percentage that is actually not Mycoplasma pneumoniae but is ...

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Abstract

The invention relates to the technical field of biology, in particular to a loop-mediated isotherm amplification (LAMP) kit for detecting mycoplasma pneumoniae (Mp). The kit contains 4 LAMP primers, the LAMP primers and an LAMP reaction solution form a detection system together, and the nucleotide sequences of the 4 LAMP primers are shown as SEQ ID No. 1-4. The kit can be used for quickly and sensitively detecting the Mp, and the lowest detection limit is 100 copies. The kit is easy to use and low in cost, the reaction result is easy to observe, and the kit has good specificity, is very suitable for disease monitoring, field emergency and detection of clinical specimens and facilitates large-range popularization and application.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a loop-mediated isothermal amplification technique (LAMP) kit for detecting mycoplasma pneumoniae. Background technique [0002] Mycoplasma pneumoniae (Mycoplasma pneumoniae, Mp) is an important pathogen that causes human respiratory tract infection, and about 10% to 40% of community-acquired pneumonia (CAP) is caused by Mp infection. Due to relatively low immune function, infants and young children are more likely to cause severe Mp infection, and even serious infections such as Mycoplasma pneumoniae encephalitis. In recent years, the incidence of pneumonia caused by Mp infection reported in China has been increasing, and the burden of disease on society has become increasingly obvious. In view of the difficulty in the isolation and culture of Mycoplasma pneumoniae, its current detection methods mainly rely on serological detection and nucleic acid detection. Since serological tec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/35
Inventor 赵飞张建中顾一心陶晓霞何利华孟凡亮肖迪
Owner ICDC CHINA CDC
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