Primer Set and Kit for Detecting Group B Streptococcus by Loop-Mediated Isothermal Amplification
A ring-mediated isothermal and streptococcal technology, applied in the direction of microorganisms, recombinant DNA technology, and microbial-based methods, can solve the problems of not being able to meet the needs of pregnancy screening, inconvenience for outpatients, and time-consuming culture and identification. Achieve the effects of low equipment and operation requirements, reduce testing costs, and improve accuracy and convenience
Active Publication Date: 2016-08-17
山东省大健康精准医疗产业技术研究院
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[0003] Bacterial culture is the standard method for detecting GBS, but this bacteria has high nutritional requirements, and it takes a long time to culture and identify, at least 2 days, and the positive rate is low, which brings inconvenience to outpatients, especially cannot meet the needs of pregnancy screening.
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[0025] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.
[0026] 1. DNA extraction
[0027] (1) DNA extraction of cultured bacteria: scrape the colony on the solid plate with a transfer loop, place it in an EP tube containing 0.2ml sterile double distilled water, put it in a boiling water bath for 8-10min, and centrifuge at 8000-12000rpm for 2-5min, Take the supernatant and transfer it to a new EP tube, which is the sample DNA, which can be directly used in the LAMP reaction.
[0028] (2) DNA extraction of clinical liquid samples: Centrifuge the liquid samples at 10000-12000rpm for 5min, remove part of the supernatant, keep the part of the precipitate, mix the precipitate with distilled water 1:1, put it in a water bath in boiling water for 8-10min, then set it at 8000-12000rpm Centrifuge for 2-5 minutes, take the supernatant and transfer it to a new EP tube, which is the sample DNA, which can be directly use...
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The invention discloses a primer set and kit for detecting group B streptococcus by a loop-mediated isothermal amplification method. The primer set comprises a pair of specific inner primers and a pair of specific outer primers. The kit comprises a plurality of reaction tubes containing 18 mu L of reaction solution, a positive control tube containing 10 mu L of GBS (group B streptococcus) DNA (deoxyribonucleic acid) and a negative control tube containing 100 mu L of sterile ultra-pure water. The kit designs primers against highly conserved and specific cAMP (cyclic adenosine monophosphate) genes in GBS genes and performs amplification and detection by adopting the loop-mediated isothermal amplification (LAMP) technology, the DNA extraction process is simple, no chemical reagents are required, the operation only needs to perform centrifugation after placing in a boiling water bath for 10min, and the influence on subsequent nucleic acid amplification caused by introduction of chemical reagents is avoided; and furthermore, the requirements on instruments, equipment and operation are low, the operation process is simplified and the detection cost is reduced.
Description
technical field [0001] The invention relates to a primer group and a kit for detecting group B streptococcus by a loop-mediated isothermal amplification method. Background technique [0002] Group B Streptococcus (GBS), also known as Streptococcus agalactiae, is a facultative anaerobic, Gram-positive streptococcus that normally resides in the vagina and intestinal tract and is an opportunistic pathogen. GBS infection causes pneumonia, urinary system infection, soft tissue infection, sepsis, etc. GBS can cause infection in newborns, pregnant women and the elderly, and is the most common pathogenic bacteria for neonatal sepsis and meningitis, with the highest infection rate in newborns. B Streptococcus infection in adults mostly occurs when the body's resistance is low. GBS resides in the mucous membranes of the urinary tract and gastrointestinal tract of the mother, and the carrier rate of pregnant women ranges from 4% to 40%, which may be passed on to their children during...
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IPC IPC(8): C12Q1/68C12Q1/14C12N15/11C12R1/46
CPCC12Q1/6844C12Q2531/119
Inventor 杨炜华汪运山裴凤艳纪明宇曹翠明张芳田娟娟
Owner 山东省大健康精准医疗产业技术研究院