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Novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatography detection kit

A streptococcus nucleic acid and immunochromatography technology, which is applied in the field of novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatography detection kits, can solve the problem of inability to effectively monitor the nucleic acid detection process, inability to set internal reference gene detection, false detection, etc. Negative results and other problems, to achieve the effect of saving detection time, high clinical generalizability, and preventing false negatives

Pending Publication Date: 2021-06-25
北京华瑞康源生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] During the entire experiment process, sample nucleic acid extraction and PCR amplification are required, and contamination or other interference factors may occur. Traditional colloidal gold has only two detection lines, which cannot be set for internal reference gene detection, and cannot effectively monitor the entire nucleic acid detection process. Cause false negative results and interfere with judgment

Method used

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  • Novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatography detection kit
  • Novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatography detection kit
  • Novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatography detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] A novel group B streptococcal nucleic acid PCR-colloidal gold immunochromatography detection kit, comprising:

[0039] (1) Amplification reaction MIX: purchased from Nanjing Novizan Biotechnology Co., Ltd. (Product No.: P211-V10.1), the reaction system includes Taq DNA polymerase, 4 kinds of dNTP and various PCR amplification reaction solutions ion.

[0040] (2) GBS detection solution: including specific labeled primers for group B Streptococcus CAMP gene, specific labeled primers for internal reference gene RNAseP and RNase Free ddH 2 O.

[0041] Group B Streptococcus amplification primers:

[0042] CX-CFB-F:5'-Bio-CTCTAGTGGCTGGTGCATTGTT-3', as shown in SEQ ID NO.1,

[0043] CX-CFB-R:5'-TAMARA-GAGTTGTCACTTGATCAGCATGT-3', as shown in SEQ ID NO.2,

[0044]The 5' end of the forward primer was labeled with biotin; the 5' end of the reverse primer was labeled with TAMARA. The primer concentration is 50 to 400 nM, preferably 200 nM.

[0045] Specific labeled primers fo...

Embodiment 2

[0072] Performance verification of the kit described in embodiment 1

[0073] (1) Sample processing

[0074] Take 200 μL sample, add 100 μL sample release agent, let stand on ice for 10 minutes, and centrifuge at 10,000 g for 2 minutes. Take 100 μL of the supernatant for use (storage at -80°C for a long time).

[0075] (2) PCR amplification

[0076] Configure the PCR amplification reaction solution according to Table 3 (20 μL for each reaction)

[0077] Table 3 PCR amplification reaction solution

[0078] components 1 reaction volume GBS test solution 7.5μL Amplification reaction MIX 12.5μL total capacity 20 μL

[0079] Aliquot the prepared PCR amplification reaction solution into 20 μL of each reaction well. Add 5 μL each of the extracted sample DNA, positive control DNA, and negative control DNA to the corresponding reaction wells, and carry out PCR amplification on the machine.

[0080] Amplification procedure:

[0081] 95°C for 3...

Embodiment 3

[0103] The kit described in Example 1 was used to detect 50 clinical samples.

[0104] (1) Sample processing

[0105] Vaginal swabs were used to collect vaginal samples from 50 pregnant women who visited the Obstetrics and Gynecology Department of Beijing Chaoyang Hospital in April 2020 for 35-37 weeks.

[0106] Key points for sample collection: 24 hours before vaginal sample collection, sexual intercourse, tub bath, vaginal examination, vaginal douche and topical medication are prohibited, so as not to affect the test results. Generally, saline-soaked cotton swabs are used to collect materials from the deep part of the vagina or the posterior part of the vaginal vault, the cervical canal, etc., and prepare 1 mL of normal saline solution of vaginal secretions for later use.

[0107] Take 200 μL sample, add 100 μL sample release agent, let stand on ice for 10 minutes, and centrifuge at 10,000 g for 2 minutes. Take 100 μL of the supernatant for use (the remaining supernatant i...

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Abstract

The invention discloses a novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatography detection kit. The kit comprises an amplification reaction MIX, a GBS detection liquid, a positive reference substance, a negative reference substance and a sample releasing agent; the GBS detection liquid comprises a specific marker primer aiming at a group B streptococcus CAMP gene, a specific marker primer of a reference gene RNAse P and RNase Free ddH2O. According to the novel group B streptococcus nucleic acid PCR-colloidal gold immunochromatography detection kit, after nucleic acid is extracted from collected cell samples, specific fragments are amplified under the action of Taq DNA polymerase, then group B streptococcus nucleic acid detection is achieved through colloidal gold immunochromatography, in the processes of sample collection, nucleic acid extraction and PCR amplification, a reference gene RNAse P is used for quality control tracking, and it is guaranteed that the whole experiment process is within a controllable range.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a novel group B streptococcal nucleic acid PCR-colloidal gold immunochromatography detection kit. Background technique [0002] Group B Streptococcus is a Gram-positive coccus, scientific name S. agalactiae (S. agalactiae), which was paid attention to by veterinary circles in the early days because it can cause bovine mastitis and seriously endanger animal husbandry. Until 1938, Fry first reported 3 cases of postpartum endocarditis deaths caused by human infection with Group B Streptococcus, confirming that Group B Streptococcus is also a human pathogen. Because the polysaccharide substance in the cell wall of the bacteria belongs to the B group in the antigen structure classification, it is also called Group B Streptococcus (Group B Streptococcus, GBS). [0003] Group B Streptococcus is an opportunistic pathogen and is internationally recognized as the leading caus...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/6804C12Q1/14C12R1/46
CPCC12Q1/689C12Q1/6804C12Q2600/166
Inventor 于超计吴文立王倩玉赵立明
Owner 北京华瑞康源生物科技发展有限公司
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