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A kind of electrochemical sensor for detecting b group streptococcus and its preparation and application

A sensor and streptococcus technology, applied in the direction of material electrochemical variables, scientific instruments, instruments, etc., can solve the problems of long time-consuming, low sensitivity, missed detection, etc.

Inactive Publication Date: 2017-04-26
CHONGQING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This culture method can intuitively interpret the results, is easy to develop and low cost, and can be used for drug sensitivity analysis, but it takes a long time, especially not suitable for pregnant women who do not know whether they have GBS infection during labor, and GBS for more than 48 hours The cultivation is difficult, the sensitivity is low, and the experimental operators need deep experience
In addition, bacterial antigen detection is easy to operate and low in cost, but it needs to be tested after cultivation, which takes a long time, and there are many serotypes of group B streptococcus, which is easy to cause missed detection and relatively low sensitivity; fully automated microbial automatic analysis Qualification systems also take a long time, are costly and expensive to equip
At present, the most widely used clinical method is real-time fluorescent PCR, which uses a designed fluorescent probe to amplify nucleic acid by PCR. ), this sequence exists in all GBS, this method and the standard bacterial culture method have reached more than 90% in the sensitivity and specificity of GBS detection, good repeatability, rapid detection, simple operation, but the operation The process requires constant temperature cycles, specialized PCR laboratories and fluorescent PCR instruments, and the need to design primers, which limits the use of this method

Method used

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  • A kind of electrochemical sensor for detecting b group streptococcus and its preparation and application
  • A kind of electrochemical sensor for detecting b group streptococcus and its preparation and application
  • A kind of electrochemical sensor for detecting b group streptococcus and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1 prepares electrochemical sensor and detects GBS

[0103] 1. Materials and methods

[0104] 1.1 Materials

[0105] Klenow Fragment(KF), T7RNA Polymerase, DEPC, RNase Inhibitor, dNTP mixture

[0106] solution and NTP mixture solution were purchased from Shanghai Shenggong. 6-Mercapto-1-hexanol (MCH), α-naphthyl

[0107] Phosphate(α-NP), streptavidin-labeled alkaline phosphatase (streptavidin-alkalinephosphatase,

[0108] ST-ALP), deionized formamide was purchased from Sigma-Aldrich (Shanghai, China). DNA purified by HPLC from

[0109] Shanghai Sangong Synthetic Co., Ltd. Clinical specimens were obtained from the First Affiliated Hospital of Chongqing Medical University.

[0110] 1.2 Testing instruments

[0111] Shanghai Chenhua CHI660D electrochemical workstation, the detection system is a three-electrode system, including an Ag / AgCl electrode as a reference electrode, a platinum wire electrode as a counter electrode, and a gold electrode with a diame...

Embodiment 2

[0134] Embodiment 2 verifies the feasibility of GBS electrochemical sensor

[0135] 1. Verification of T / E / GBS, polymeric extension products and transcription amplification products

[0136] The T / E / GBS obtained in Example 1, the polymerization extension product and the transcription amplification product are verified by agarose gel electrophoresis and electrochemical process, such as figure 1 Sections 3, 4, and 5 in A show that T (template probe), E (extension probe) and GBS can be combined, and can be polymerized and extended under the action of KF enzyme, and can be transcribed and amplified under the action of T7RNA Polymerase. Nucleic acid fragments of approximately 40 bp. figure 1 It can also be seen in B that the ideal electrochemical signal can only be obtained when KF and T7RNA Polymerase are added at the same time.

[0137] Such as figure 1 A:

[0138] Band 1 is a 500bp Marker;

[0139] Stripe 2 is GBS;

[0140] Band 3 is T / E;

[0141] Band 4 is T / E / GBS;

[0...

Embodiment 3

[0150] The optimization of embodiment 3GBS electrochemical sensor and service conditions thereof

[0151] We also analyzed several important conditions during the experiment, namely, the number of T / E base pairings, the optimization of transcription amplification mediated by defective T-shaped structures, the hybridization of transcription amplification products and DP The temperature of the polymerase, the time of polymerization extension, the concentration of T7RNA Polymerase, and the reaction time of the transcription amplification system were further optimized. For each optimized condition, at least five points were selected from low concentration to high concentration. A series of experiments.

[0152] 1. In order to investigate the influence of the number of T / E base pairings on the detection results of the GBS electrochemical sensor, this experiment used different numbers of base pairings to construct the GBS electrochemical sensor. Such as figure 2 It can be seen th...

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Abstract

The invention relates to the field of nucleic acid detection, and particularly relates to an electrochemical sensor for detecting group B streptococcus (GBS) and preparation and an application thereof. The sensor comprises a working electrode, a reference electrode and a counter electrode, wherein the working electrode is obtained by fixing a capturing probe on the surface of a substrate electrode, namely a gold electrode, and the sensor further comprises a template probe, an extension probe and a detection probe which are matched with the capturing probe. The sensor can be used for detecting GBS sensitively, quickly and specifically.

Description

technical field [0001] The invention relates to the field of nucleic acid detection, in particular to an electrochemical sensor for detecting group B streptococcus and its preparation and application. Background technique [0002] Group B Streptococcus (GBS) is a Gram-positive coccus that normally resides in the vagina and rectum of the human body. It is an opportunistic pathogen that can cause severe sepsis and neonatal meningitis infection. It is also one of the four major causes of maternal death. The infection rate of GBS in pregnant women is as high as 20%-40%, among which the proportion of vertical transmission from mother to child is as high as 75% and nearly 1% of infected babies will develop early-onset GBS sepsis. In view of the high infectivity and harmfulness of GBS mentioned above, it is particularly necessary to carry out prenatal screening for pregnant women. According to the GBS prevention guidelines issued by the US Centers for Disease Control and Preventi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/26G01N27/327
Inventor 程伟丁世家袁睿颜玉蓉张玉洪张晔
Owner CHONGQING MEDICAL UNIVERSITY
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