Condition optimizing method for living microbe quantitative detection technology PMA-qPCR (propidium monoazide-quantitative polymerase chain reaction)
An optimization method and quantitative detection technology, applied in the field of microbial detection, can solve the problems of high cost, long time, high operation intensity, etc., and achieve the effect of optimal PMA concentration and exposure time, and low cost.
Inactive Publication Date: 2015-02-04
FUJIAN MEDICAL UNIV
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Problems solved by technology
[0008] In order to overcome the shortcomings of the existing PMA-qPCR technology in the quantitative detection of living bacteria, the condition optimization link is high in cost, time-consuming and high in operation intensity, the present invention provides A PMA-qPCR condition optimization method based on flow cytometry (Flow Cytometry, FCM), which can directly perform FCM analysis on PMA-treated bacterial cells. Quickly and easily obtain optimal PMA concentration and exposure time at low cost
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Embodiment 1
[0047] Embodiment 1: In this embodiment, Escherichia coli ( Escherichia coli ) Top 10 strains were used as the research object. For the quantitative detection of live bacteria using PMA-qPCR, the usual method based on qPCR and the new method based on FCM were used to optimize the optimal PMA treatment concentration and exposure time. The concrete steps and results of this embodiment are as follows:
[0048] 1. Preparation of E. coli suspension
[0049] Viable bacterial suspension: transfer the Escherichia coli stored in the slant medium to the LB shaker medium, culture at a constant temperature at 37°C for about 12 hours, take 30mL of uniform bacterial solution in a 50mL sterilized centrifuge tube, and centrifuge at 10000rpm for 3 minutes Discard the supernatant, suspend and mix the precipitate with sterilized normal saline, let it stand for 1 min, then centrifuge at 10,000 rpm for 3 min, discard the supernatant to collect bacteria, add 30 mL of sterilized normal saline to s...
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The invention belongs to the field of microbiological detection, relates to a condition optimizing method of a detection technology and particularly relates to a condition optimizing method of a living microbe quantitative detection technology, namely PMA-qPCR (propidium monoazide-quantitative polymerase chain reaction). In order to overcome the defects that in the quantitative detection of living microbes, an existing PMA-qPCR technology is high in cost of condition optimizing link, long in time consumption and great in operation strength, the invention provides the condition optimizing method of the living microbe quantitative detection technology PMA-qPCR based on flow cytometry (FCM). The condition optimizing method can be used for directly performing FCM analysis on PMA-treated bacterial cells; the operations of genome extraction and qPCR amplification are cancelled, and therefore, the optimal PMA concentration and the optimal exposure time can be quickly and conveniently obtained at very low cost. For PMA treatment condition optimizing, the method disclosed by the invention has the advantages of quickness, simplicity and low cost.
Description
technical field [0001] The invention belongs to the field of microorganism detection, and relates to a method for optimizing conditions of a detection technology, in particular to a method for optimizing conditions of a live bacteria quantitative detection technology: PMA-qPCR. Background technique [0002] Real-time fluorescent quantitative PCR (qPCR) technology is the gold standard for the quantification of specific nucleic acid fragments, and is currently the most widely used and effective means of quantifying nucleic acid molecules. Considering that the number of bacteria is directly proportional to the specific nucleic acid molecules they contain (such as 16S rDNA), qPCR technology is also used in the field of bacterial quantitative detection. However, in the actual microbial ecosystem, there are living bacteria and dead bacteria; although the dead bacteria have died, their DNA will exist for a long time, so qPCR quantitatively reflects the results of the total bacteria...
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Login to View More IPC IPC(8): C12Q1/68C12Q1/06
CPCC12Q1/6851C12Q1/689C12Q2565/626C12Q2545/114
Inventor 黄志清陈智超陈强陈艳黎巧连
Owner FUJIAN MEDICAL UNIV