Kit for synchronous detection of twenty three respiratory pathogens and detection method thereof

A simultaneous detection and respiratory technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., to achieve the effect of simple operation, fast detection speed and high sensitivity

Active Publication Date: 2019-12-17
深圳市百迈生命科学有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report in China that completes reverse transcripti...

Method used

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  • Kit for synchronous detection of twenty three respiratory pathogens and detection method thereof
  • Kit for synchronous detection of twenty three respiratory pathogens and detection method thereof
  • Kit for synchronous detection of twenty three respiratory pathogens and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, a kit for simultaneously detecting 23 kinds of respiratory pathogens and a detection method thereof.

[0044] 1. Primer set combination:

[0045] Assemble the 5X primer mixture according to the sequence and concentration in the table below. The primers were synthesized by Shanghai Sangong, purified by HPLC, and inspected by CE quality. The purity is above 95%:

[0046]

[0047]

[0048] 2. 2.5X Reaction MIX combination:

[0049] 2.5X Reaction Mix (Reaction mix) contains: 125mM Tricine-KOH (pH 8.8, 25°C), 100mM KCl, 40mM (NH4)2SO4, 6mM MgCl2, 1.2mM dNTPs, 2.5mM dTT, 2.5mg / ml BSA, 1M Betaine , 1% Tween 20.

[0050] 3. Sample source and extraction:

[0051] 23 kinds of respiratory pathogenic bacteria, after identification by culture method, took 500CFU of various bacterial liquids, samples were donated by a provincial CDC, inactivated and mixed into test samples, the mixed samples were diluted 10 times and 100 times respectively, and tested by QIAamp...

Embodiment 2

[0060] Embodiment 2, a kit for simultaneously detecting 23 kinds of respiratory pathogens and a detection method thereof.

[0061] 1. The three sample types for sample extraction are 1. Nasopharyngeal swab, 2. Pharyngeal swab, 3. Nasopharyngeal lotion, extracted by QIAamp MinElute Virus Spin Kit (Product No. 57704, Qiagen) and operated according to the instructions. Finally, it was eluted with 50 microliters of Buffer AVE.

[0062] 2. According to the configured primer set and reaction mix in Example 1, carry out PCR reaction with the sample loading and amplification procedures in Example 1.

[0063] 3. Electrophoresis detection, a sample loading mixture composed of deionized formamide and molecular weight internal standard (ABI LIZ-120, USA) [(0.5 μL molecular weight internal standard + 10 μL deionized formamide)×(number of samples)]. Mix 9 μL loading mixture with 1 μL amplification product, and centrifuge briefly to collect the liquid at the bottom of the centrifuge tube. ...

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Abstract

The invention discloses a kit for synchronous detection of twenty three respiratory pathogens and a detection method thereof. The kit comprises: a transcription and amplification primer, 2.5 x Reaction MIX, DEPC water, a hot-start tap enzyme and a reverse transcriptase, wherein the transcription and amplification primer includes primer sequences of twenty three respiratory pathogens including 17 RNA viruses, 2 DNA viruses and 4 respiratory tract pathogenic bacteria, and the gene sequences are as shown in SEQ ID NO.1-NO.44. After pathogen samples are extracted, transcription and amplification are completed through single-tube reaction of the kit, capillary electrophoresis detection is performed, and pathogen categories are determined through software analysis of results. The technical scheme has the advantages of strong specificity, high sensitivity, fast detection speed, completion of transcription amplification by a single tube, and simple and convenient operation.

Description

technical field [0001] The invention relates to a detection kit for respiratory pathogens and a detection method thereof, in particular to a kit for synchronous detection of 23 respiratory pathogens based on multiple fluorescent PCR capillary electrophoresis and a detection method thereof. Background technique [0002] Respiratory tract infections have become increasingly serious in recent years, especially among children and adolescents. The pathogens that cause respiratory tract infection mainly include microorganisms such as viruses, bacteria, mycoplasma, and chlamydia. One pathogen can cause multiple clinical manifestations, and the same clinical manifestation can be caused by multiple pathogens. Due to the complex and unobvious clinical symptoms caused by these atypical pathogens, patients are often ignored or misdiagnosed, and it is difficult to treat pathogens clinically, which may aggravate the condition of some patients or cause the abuse of antibacterial drugs. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12Q1/686C12Q1/04C12N15/11C12R1/01
CPCC12Q1/701C12Q1/689C12Q1/686C12Q2600/16C12Q2563/107C12Q2537/143Y02A50/30
Inventor 车雷车力王宇鹏
Owner 深圳市百迈生命科学有限公司
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