Anaerobe liquid-phase chip inspection method

A liquid-phase chip detection and chip detection technology, applied in the field of detection, can solve the problems of inability to meet the needs of early clinical, sensitive and specific diagnosis, time-consuming isolation and identification of bacterial species, and low detection rate, etc. The effect of treatment, reducing medical expenses, reducing medical expenses

Pending Publication Date: 2020-06-05
东莞市厚街医院
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  • Abstract
  • Description
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Problems solved by technology

[0003] The detection of anaerobic bacteria is mainly based on the traditional bacterial culture and identification based on their phenotypic characteristics and biochemical reactions. Aerobic bacteria grow slowly and are often mixed with other pathogens. The entire detection cycle is as long as 3-7 days. The difficulty of identification leads to low accuracy and high false negative rate. Moreover, the whole process requires a special anaerobic environment and expensive anaerobic equipment. Bacteria culture equipment, which limits the development of many laboratories in China, is far from meeting the needs of early clinical, sensitive and specific diagnosis

Method used

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  • Anaerobe liquid-phase chip inspection method
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  • Anaerobe liquid-phase chip inspection method

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Embodiment 1

[0051] A liquid phase chip detection method for anaerobic bacteria, comprising the following steps:

[0052] Step A: Design primers and probes;

[0053] Step B: establishment of multiple RT-PCR reaction system;

[0054] Step C: establishment of a liquid phase chip detection system;

[0055] Step D: Detection.

[0056] In the step A, the specific steps of designing primers are as follows: select the specific sequence of the 16S rDNA gene of nine kinds of anaerobic bacteria to design the upstream and downstream primers of various bacteria, the 5' end of the downstream primer is modified with biotin, and the nine kinds of anaerobic bacteria The primer sequences of bacteria are as follows:

[0057]

[0058]

[0059] In the step A, the specific steps of designing the probe are as follows: find out the specific sequence within the species by comparison and design the probe as the specific region for screening, and modify the 5' end with amino and C12 during synthesis, and u...

Embodiment 2

[0090] This embodiment is to analyze and verify the specificity, sensitivity and repeatability of the liquid chip of the above-mentioned embodiment 1:

[0091] 1. Specificity evaluation: 9 kinds of anaerobic bacteria were detected to ensure that the species would not cross each other, and each probe on the chip was evaluated for specificity. The fluorescent signal values ​​of specificity detection are shown in Table 3.

[0092] Table 3. Specific detection fluorescence signal value (MFI) of each strain

[0093]

[0094]

[0095] 2. Sensitivity analysis: measure the genome concentration of each bacteria by ultraviolet spectrophotometer, and dilute to 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, 1pg / μL, 100fg / μL, 10fg / μL, 1fg / μL, PCR amplification of the genome, chip hybridization, and detection on the machine. Among them, the detection sensitivity of Bacteroides fragilis, Fusobacterium nucleatum, Veillonella parvum, Actinomyces clostrii, Clostridium perfringens, Porphyr...

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Abstract

The invention relates to the technical field of detection, in particular to an anaerobe liquid-phase chip inspection method. The detection method comprises the following steps of step A, designing a primer and a probe; step B, establishing a multiple RT-PCR reaction system; step C, establishing a liquid-phase chip detection system; and step D, performing detection. The detection method is based ondesign basis of 16S rDNA molecule subtypes, through combination of a multiplex PCR reaction, a Luminex technique is used as a platform, the liquid-phase chip method is established, and besides, the method is used for detecting 9 anaerobe nucleic acids which are common in clinical application and is convenient to operate, free from requirement for viable bacteria, high in flux, high in sensitivity, high in speed, high in specificity and low in cost. Timely and accurate information can be provided for clinical application, medical resources are saved, the medical treatment charge is effectivelyreduced, early diagnosis of anaerobe is favorable for early treatment of clinical application, the hospitalization period of patients is effectively shortened, the medical treatment charge of the patients is reduced, and joint medical resources are saved.

Description

technical field [0001] The invention relates to the technical field of detection, in particular to a liquid-phase chip detection method for anaerobic bacteria. Background technique [0002] Anaerobic bacteria are part of the normal flora of the human body and are widely distributed in various parts of the human body. When the body's systemic or local resistance is reduced, the colonization position or amount of normal flora stored in the body changes, which will lead to the development of anaerobic bacteria. Endogenous infection can not only cause severe inflammation, abscess, and soft tissue necrosis, but also systemic factors that can cause infection are mostly complex diseases, such as diabetes, immunosuppression, tumors, and liver cirrhosis. Local factors are mostly limitations, such as mucous membrane injury, local tissue ischemia, hypoxia, shock, edema, etc., which are conducive to the occurrence and development of anaerobic infection. Studies have pointed out that th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6837C12Q1/04
CPCC12Q1/689C12Q1/6837C12Q2600/16C12Q2600/166C12Q2537/143C12Q2545/113C12Q2531/107C12Q2565/501Y02A50/30
Inventor 付文金陈梅莲邓任堂张露赖丽莎
Owner 东莞市厚街医院
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