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Method for detecting genital tract mycoplasmas based on loop-mediated isothermal amplification and micro-fluidic chip

A microfluidic chip, ring-mediated constant temperature technology, applied in the direction of micro-organism-based methods, micro-organism measurement/inspection, biochemical equipment and methods, etc., to achieve the effect of high overall coincidence rate, avoiding subjective factors, and good sensitivity

Pending Publication Date: 2020-06-19
重庆市第四人民医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the detection methods for Mycoplasma genitalium are becoming more and more abundant, from the perspective of technical maturity and experimental cost, there is still no perfect technology that can be put into clinical diagnosis.

Method used

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  • Method for detecting genital tract mycoplasmas based on loop-mediated isothermal amplification and micro-fluidic chip
  • Method for detecting genital tract mycoplasmas based on loop-mediated isothermal amplification and micro-fluidic chip
  • Method for detecting genital tract mycoplasmas based on loop-mediated isothermal amplification and micro-fluidic chip

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 is used for the design and preparation of mycoplasma genitalium detection primers

[0032] The functional gene sequences of four mycoplasmas Uu, Up, Mh, and Mg were obtained from the Genebank public database, multiple sequence alignments were performed with Muscle, and consensus sequences were obtained by combining tools. The consensus sequence is used as input, combined with tools to cut primer sequences that meet the requirements. The primers should meet the following conditions: 1) 35%<GC content<65%; 2) The primers will not form a hairpin structure, and the reverse complementary sequence should not be longer than 5mer; 3) The primer sequence cannot have more than 5 consecutive single base repeats; 4) There will be no dimer formation between the primers. Use the cut primer sequence to perform blast comparison, combine with tools to obtain preliminary primer screening results, use NT and other public databases for further primer screening and specificity det...

Embodiment 2

[0035] Example 2 Detection of Mycoplasma genitalium based on loop-mediated constant temperature amplification and microfluidic chip

[0036] (1) Design and manufacture of disc chips; for the structure of disc chips, see figure 1 , each chip can detect 3 samples (I, II, III), each sample contains 8 reaction pools, numbered counterclockwise, respectively 4 mycoplasma detection primers (Uu, Up, Mg, Mh), 1 Positive control (PC), 1 negative control (NC), 2 blank controls (BC). Each chip embeds and fixes a set of primers in a specific reaction pool for the amplification and detection of a nucleic acid target sequence. The primer layout corresponding to each reaction pool is shown in Table 2.

[0037] Table 2 Microfluidic chip detection index layout

[0038] Reaction pool Detection Indicator Reaction pool Detection Indicator I-1 BC II-5 Mg I-2 PC II-6 mh I-3 u II-7 NC I-4 up II-8 BC I-5 Mg III-1 BC I-6 mh III-2...

Embodiment 3

[0043] Example 3 Verification of the accuracy of the microfluidic chip

[0044] The isothermal amplification microfluidic chip prepared in Example 2 was used to verify the accuracy. The standard strains of 4 kinds of mycoplasma were used as positive reference products, and the nucleic acid analyzer chip was used to detect them to verify the accuracy of the chip for sample detection. Operation steps with reference to embodiment 2.

[0045] The chip can correctly detect 4 kinds of mycoplasma positive reference products, such as figure 2 shown. The chip was detected for 50 minutes in total, among which the peak time of the Uu amplification curve was 10.04 minutes, the peak time of the Up amplification curve was 11.58 minutes, the peak time of the Mg amplification curve was 17.18 minutes, and the peak time of the Mh amplification curve was 25.42 minutes .

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Abstract

The invention discloses a method for detecting genital tract mycoplasmas based on loop-mediated isothermal amplification and a micro-fluidic chip and belongs to the technical field of pathogen detection and diagnosis. The method comprises the following steps: performing primer designing; manufacturing the micro-fluidic chip; preparing an isothermal amplification reaction system; supplying a sampleto the chip; and performing nucleic acid amplification, and judging and reading results. An isothermal amplification technique and a micro-fluidic chip technique are used in the method, polymerase with a chain replacement function is reacted under an isothermal condition of 63 DEG C, real-time fluorescence detection is implemented by using a fluorescence dye mixing method, and a sample with amplification positive can generate an ''S''-shaped amplification curve similar to real-time fluorescence. The micro-fluidic chip successfully prepared for four micro-fluidic chips has good sensitivity, specificity and accuracy, and the whole detection process can be completed within 1 hour. The minimum detection limits of Uu, Mg and Mh are 1000 copies / [mu] L, the minimum detection limit of Up is 5000copies / [mu] L, and the method has wide application prospects in clinical use.

Description

technical field [0001] The invention relates to the technical field of pathogen detection and diagnosis, in particular to a method for detecting mycoplasma of the genital tract based on a loop-mediated constant temperature amplification and a microfluidic chip. Background technique [0002] Mycoplasma is a class of smallest prokaryotic microorganisms whose size is between bacteria and viruses, lacks cell walls, is highly pleomorphic, can pass through bacteria filters, and can be cultured and proliferated in artificial media. The mycoplasmas that have been found to be associated with reproductive tract infections mainly include Ureaplasma urealyticum (Uu), Ureaplasma parvum (Up), Mycoplasma hominis (Mh), Mycoplasma genitalium (Mg ). The liquid culture method is the main detection method for Uu and Mh in the former domestic medical institutions. This method has the advantages of simple operation, fast and economical, can be observed with the naked eye, and simultaneously cond...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/04C12R1/35
CPCC12Q1/689C12Q1/6844C12Q2531/119C12Q2565/629
Inventor 张薇
Owner 重庆市第四人民医院
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