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Mycoplasma nucleic acid isothermal amplification method

A technology of mycoplasma and amplification reaction, which is applied in the direction of biochemical equipment and methods, DNA/RNA fragments, microorganism measurement/inspection, etc. It can solve the problems that there are no research reports on real-time fluorescent nucleic acid constant temperature amplification detection technology of mycoplasma genitalium

Active Publication Date: 2015-02-11
SHANGHAI RENDU BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the problems faced by the application of SAT technology in the detection of different types of viruses are different, and it is necessary to specifically analyze the characteristics of the virus for special design.
At present, there is no research report on the real-time fluorescent nucleic acid constant temperature amplification detection technology for Mycoplasma genitalium (MG) and Mycoplasma pneumoniae (MP)

Method used

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  • Mycoplasma nucleic acid isothermal amplification method
  • Mycoplasma nucleic acid isothermal amplification method
  • Mycoplasma nucleic acid isothermal amplification method

Examples

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Embodiment 1

[0249] Embodiment 1 is used for real-time fluorescent nucleic acid constant temperature amplification to detect the design of special primers and probes of Mycoplasma genitalium (MG)

[0250] The present invention selects no secondary structure and a highly conserved segment in the MG virus 16SRNA gene as the amplified target sequence region (its nucleotide sequence is shown in SEQ ID NO: 1), and according to the principle of primer probe design, DNAStar, DNAMAN Software and artificially designed special primers and probe sequences for real-time fluorescent nucleic acid constant temperature amplification detection of Mycoplasma genitalium (MG), and obtained the following specific sequences:

[0251] (1) a capture probe (TCO, Target Capture Oligo) that can specifically bind to the target nucleic acid (MG RNA) sequence of Mycoplasma genitalium (MG), the nucleotide sequence of the capture probe is: 5'TGCACACCGGATGGCCAATCCAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3' (SEQ ID No.: 2);

[0...

Embodiment 2

[0258] Embodiment 2 is used for the design of special primers and probes of real-time fluorescent nucleic acid constant temperature amplification detection Mycoplasma pneumoniae (MP)

[0259] The present invention selects no secondary structure and a highly conserved segment in the MP virus 16SRNA gene as the amplified target sequence region (its nucleotide sequence is shown in SEQ ID NO: 10), and according to the principle of primer probe design, DNAStar, DNAMAN Software and artificially designed special primers and probe sequences for real-time fluorescent nucleic acid constant temperature amplification to detect Mycoplasma pneumoniae (MP), and obtained the following specific sequences:

[0260] (1) a capture probe (TCO, Target Capture Oligo) that can specifically bind to the target nucleic acid (MP RNA) sequence of Mycoplasma pneumoniae (MP), the nucleotide sequence of the capture probe is: 5'TGCACACCGGATGGCCAATCCAATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3' (SEQ ID No.: 2);

[026...

Embodiment 3

[0267] The real-time fluorescent nucleic acid constant temperature amplification detection kit of embodiment 3 Mycoplasma genitalium (MG)

[0268] Using the special primers and probes provided in Example 1, the real-time fluorescent nucleic acid constant temperature amplification detection kit for Mycoplasma genitalium (MG) of the present invention was obtained. The kit contains components such as capture probe (TCO, Target Capture Oligo), T7 primer, nT7 primer, MG detection probe, internal standard detection probe, internal standard, M-MLV reverse transcriptase and T7 RNA polymerase .

[0269] The capture probe exists in the nucleic acid extraction solution, the T7 primer, nT7 primer, MG detection probe, and internal standard detection probe exist in the MG detection solution, and the M-MLV reverse transcriptase and T7 RNA polymerase It exists in the SAT enzyme solution. Specifically, the kit is divided into box A (specimen processing unit) stored at 2-30°C and box B (nuclei...

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Abstract

The invention discloses a mycoplasma nucleic acid isothermal amplification method. In particular, the mycoplasma nucleic acid isothermal amplification method includes the step that amplification is carried out in a reaction system, wherein the reaction system comprises specific primer pairs for amplifying mycoplasmas, and primers can be used for amplifying amplification products corresponding to mycoplasma feature sequences from a detection sample with an extra-low mycoplasma copy number. By means of the mycoplasma nucleic acid isothermal amplification method, the to-be-detected sample containing mycoplasma RNA can be rapidly amplified with high specificity, high flexibility and low pollution; and the mycoplasma nucleic acid isothermal amplification method is particularly suitable for amplification and detection of the to-be-detected sample containing mycoplasma genitalium MG and / or mycoplasma pneumoniae MP. The mycoplasma nucleic acid isothermal amplification method has the beneficial effects of being high in detection efficiency and high in accuracy.

Description

technical field [0001] The invention relates to the technical field of biological detection of microorganisms, in particular to primers, probes and related kits used in the real-time fluorescent nucleic acid constant temperature amplification detection of mycoplasma by combining specific target capture technology and real-time fluorescent nucleic acid constant temperature amplification detection technology. Background technique [0002] Mycoplasma is a kind of prokaryotic microorganism between bacteria and virus, which lacks cell wall and can replicate independently. Parasites in the genitourinary tract of humans and other primates rely on highly specific parasitic mechanisms including unique apical organelle transplantation and invasion into human cells. [0003] In recent years, a large amount of epidemiological data suggest that Mycoplasma genitalium MG is a common pathogen of human genitourinary tract, is related to many non-gonococcal urethritis (NGU), prostatitis and p...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6844C12Q1/689C12Q2561/113C12Q2563/107C12Q2565/519
Inventor 王敏马道亮汤嘉维于明辉居金良
Owner SHANGHAI RENDU BIOTECH
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