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Primer probe combination and detection kit for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus

A technology for Mycoplasma pneumoniae and Chlamydia pneumoniae, which is applied in the biological field, can solve problems such as unreasonable involvement of primers and probes, and achieve the effects of good specificity, cost saving and sensitive identification.

Pending Publication Date: 2021-02-26
AUTOBIO DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, at present, there are unreasonable phenomena in the selection of gene fragments identified for MP, CP and ADV and the involvement of primers and probes, resulting in the inability of most detection reagents to achieve accurate, rapid and sensitive detection

Method used

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  • Primer probe combination and detection kit for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus
  • Primer probe combination and detection kit for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus
  • Primer probe combination and detection kit for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Preparation of mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus triple nucleic acid detection kit

[0057] The primer and probe sequences in the kit are shown in Table 1 below:

[0058] Table 1 Primer, probe sequence

[0059]

[0060]

[0061] The kit also includes: 10mM dNTPs, 1U / μL UDG enzyme, 5U / μL Taq enzyme, 50mM MgCl 2 . The kit also includes a negative control (sterile water) and a positive control (artificially synthesized at a concentration of 1×10 6 Copies / mL of pseudovirus).

Embodiment 2

[0062] The detection method of embodiment 2 kit of the present invention

[0063] Detection method of the present invention is Real time RT-PCR, and Real Time RT-PCR reaction process is:

[0064] The composition of each detection system is shown in Table 2:

[0065] Table 2 Detection system

[0066]

[0067]

[0068] Fluorescence detection channel selection:

[0069] (1) Select the FAM channel (ReporTer: FAM, QuenCher: none) to detect Mycoplasma pneumoniae; (2) Select the CY5 channel (ReporTer: CY5, QuenCher: none) to detect Chlamydia pneumoniae; (3) Select the ROX channel (ReporTer: ROX , QuenCher: none), detect adenovirus; (4) select HEX channel, detect internal standard; (5 reference fluorescence (PAssiveReferenCe) is set to none. Fluorescent quantitative real-time reaction conditions are shown in Table 3 below.

[0070] Table 3: Fluorescent quantitative real-time PCR reaction conditions

[0071]

[0072] After the reaction, the instrument automatically saves t...

Embodiment 3

[0084] The feasibility test of embodiment 3 kit of the present invention

[0085] 1. Sensitivity evaluation test

[0086] (1) Preparation of triple nucleic acid detection reagents for Mycoplasma pneumoniae, Chlamydia pneumoniae and adenovirus: The triple nucleic acid detection reagent for Mycoplasma pneumoniae, Chlamydia pneumoniae and adenovirus was prepared by adopting the method in Example 1.

[0087] (2) Pathogen sample extraction

[0088] Use a pipette to mix the 5 different concentrations of mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus human throat swab eluate in the tube, take out 100 μl into a new centrifuge tube, centrifuge at 12000rpm for 5min, and carefully discard the supernatant; Add 200 μL of bacterial lysate (from Beijing Biolab Technology Co., Ltd.) to the precipitate, mix well, bathe in water at 100°C for 5 minutes, and centrifuge at 10,000 rpm for 5 minutes for later use.

[0089] (3) Sample detection

[0090] Add 25 μL of the processed speci...

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Abstract

The invention relates to the technical field of biology, in particular to a primer probe combination and a detection kit for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus. The primer probe combination comprises a specific primer pair of mycoplasma pneumoniae shown in SEQ ID NO: 1-2, a specific probe of mycoplasma pneumoniae shown in SEQ ID NO:3, a specific primer pair of chlamydia pneumoniae shown in SEQ ID NO: 4-5, a specific probe of chlamydia pneumoniae shown in SEQ ID NO:6, a specific primer pair of adenovirus shown in SEQ ID NO: 7-8 and a specific probe of adenovirus shown in SEQ ID NO: 9. The primers and the probes have good specificity, and rapid, accurate and sensitive identification of MP, CP and ADV can be achieved in combination with a real time PCR detection method.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a combination of primers and probes and a detection kit for detecting mycoplasma pneumoniae, chlamydia pneumoniae and adenovirus. Background technique [0002] Mycoplasma pneumoniae (MP) is the causative agent of human mycoplasma pneumonia. The pathological changes of mycoplasma pneumonia are mainly interstitial pneumonia, sometimes complicated by bronchial pneumonia, which is called primary atypical pneumonia. It is mainly transmitted by droplets, with an incubation period of 2 to 3 weeks, and the highest incidence rate is among adolescents. The clinical symptoms are mild, or even asymptomatic. If there are, there are only general respiratory symptoms such as headache, sore throat, fever, and cough, but there are also reports of individual deaths. It can happen all year round, but mostly in autumn and winter. [0003] According to genetic and biological characteristics, Chlamydia...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/6848C12Q1/04C12N15/11C12R1/35C12R1/01C12R1/93
CPCC12Q1/701C12Q1/689C12Q1/6848C12Q2600/166C12Q2531/113C12Q2521/531C12Q2545/101C12Q2563/107Y02A50/30
Inventor 兰定云高利飞李进福麦艳娜李振红王玮付光宇
Owner AUTOBIO DIAGNOSTICS CO LTD
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