Quantum dot nucleic acid detection kit and method for simultaneously detecting 24 respiratory tract pathogens

A detection kit and quantum dot technology, applied in the field of biomedicine, can solve problems such as incomplete species of respiratory pathogens

Active Publication Date: 2020-07-10
杭州千基生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The types of respiratory pathogenic bacteria publicly detected in the prior art are incomplete, and only include some common viruses or bacteria or mycoplasma or chlamydia, etc. The present invention pr

Method used

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  • Quantum dot nucleic acid detection kit and method for simultaneously detecting 24 respiratory tract pathogens
  • Quantum dot nucleic acid detection kit and method for simultaneously detecting 24 respiratory tract pathogens
  • Quantum dot nucleic acid detection kit and method for simultaneously detecting 24 respiratory tract pathogens

Examples

Experimental program
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Effect test

Embodiment example 1

[0139] Implementation Case 1: Preparation and Use of the Kit for Quantum Dot Nucleic Acid Detection of Respiratory Pathogenic Bacteria in the Present Invention

[0140] 1. Quantum dot nucleic acid detection principle: Molecular hybridization is carried out between biotin-labeled nucleic acid amplification products and probes on the detection membrane strip, and then biotin is combined with streptavidin-coupled quantum dots to detect membrane strips. The strips are detected by a fluorescence detection instrument for the presence or absence of light signals at each site to determine whether the probe hybridizes with the nucleic acid product, thereby determining whether the sample contains the relevant target nucleic acid.

[0141] The capture probe is labeled with an amino group at the 3' end or 5' end of the oligonucleotide single-stranded DNA, and there is an intermediate arm between the amino group and the oligonucleotide single-stranded DNA, and the intermediate arm is a fatt...

Embodiment 2

[0273] The present invention is used for the sensitivity verification of 24 kinds of respiratory pathogenic bacteria quantum dot nucleic acid detection kits:

[0274] According to the reaction system in Example 1, 21 ul were respectively dispensed, and 4 ul of DNA / RNA was added to each reaction system, with a concentration of 5000 copies / ml and 500 copies / ml.

[0275] RT-PCR amplification program carries out PCR amplification according to the program described in embodiment 1, specifically as follows:

[0276]

[0277] The detection process of the quantum dot gene chip is carried out according to the kit use process in Example 1 for relevant detection. The test results of the three-tube reaction can be found in figure 1 , figure 2 , image 3 . The results of fluorescence detection show that the detection target sensitivity of the kit can reach 500copies / ml.

Embodiment example 3

[0278] Implementation Case 3: Throat swab clinical sample detection

[0279] Collect 12 cases of throat swab samples suspected of respiratory tract infection according to clinical microbiology sample collection specifications, and use the virus nucleic acid extraction kit produced by Hangzhou Qianji Biotechnology Co., Ltd. to extract nucleic acid, and then use the reaction system in Example 1 to perform nucleic acid extraction Sample detection, specific amplification process and detection process refer to Example 1. The specific test results are as follows:

[0280] sample number Example test results Sequencing results 1 ND ND 2 H1N1 (2009) H1N1 (2009) 3 H1N1 (2009) H1N1 (2009) 4 229E 229E 5 HKU1 HKU1 6 ND ND 7 ND ND 8 RSVB RSVB 9 H1N1 (2009) H1N1 (2009) 10 RSVA / HKU1 RSVA / HKU1 11 H1N1 (2009) H1N1 (2009) 12 ND ND

[0281] Note: ND is marked as the detection of releva...

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Abstract

The invention discloses a quantum dot nucleic acid detection kit and method for simultaneously detecting 24 respiratory tract pathogens. The kit comprises reaction solutions I-V, a positive control, an internal label and a negative control. The reaction solutions I-III comprise a respiratory tract pathogen detection primer, dN(U)TP, UDG, a PCR buffer system and an RNA enzyme inhibitor; the reaction solution IV comprises DNA polymerase; the reaction solution V comprises reverse transcriptase and DNA polymerase; the positive control comprises influenza A viruses, coronavirus OC43 pseudoviruses and mycoplasma pneumoniae recombinant plasmids; the internal label comprises exogenous fragment pseudoviruses free of target genes; the negative control comprises human genomic DNA; a fluorescence detection solution comprises a streptavidin-coupled quantum dot; a denaturant comprises an alkali solution; a neutral agent comprises an acid solution; and a membrane strip comprises a nylon membrane comprising a coupling-specific detection probe. The 24 common respiratory tract infection pathogens can be quickly, sensitively and specifically detected, and the occurrence of false negatives can be effectively monitored.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a quantum dot nucleic acid detection kit and method for simultaneously detecting 24 kinds of respiratory pathogenic bacteria. Background technique [0002] Respiratory tract infection (Respiratory tract infection, RTI) is the most common type of disease in humans, which can occur in any gender, age and region, and is one of the most important causes of morbidity and mortality worldwide. The clinical symptoms and signs caused by respiratory tract infection are relatively similar, and its clinical manifestations are mainly rhinitis, pharyngitis, laryngitis, tonsillitis and other symptoms, and severe cases can cause tracheitis, bronchitis and pneumonia, etc. The treatment methods, efficacy and course of the disease also vary. It has been proven that most respiratory diseases are caused by pathogens other than bacteria, among which respiratory viruses are the most co...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/689C12N15/70C12N1/21
CPCC12Q1/701C12Q1/689C12N15/70C12Q2600/16Y02A50/30
Inventor 尹华立裘惠良
Owner 杭州千基生物科技有限公司
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