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An identification method and microorganism technology, applied in the field of microorganism identification, to achieve the effect of high sensitivity and resolution
Inactive Publication Date: 2009-02-11
ZHEJIANG UNIV
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Embodiment 1
[0068] Embodiment 1: Extract is the high-throughput microbial identification method of DNA or RNA
[0069] (1) Sampling
[0070] DNA was extracted from the cultured bacteria, and when the OD value of the culture reached about 0.2, about 50 microliters of the culture were transferred to 300 microliters of ATL buffer (Qiagen Company) for pretreatment.
[0071] (2) Extraction of genetic material
[0072] Use Qiagen's DNeasy Tissue kit for gene extraction, and follow the instructions.
[0073] (3) Amplification
[0074] The PCR conditions during the bacteria detection process are: 50 microliters of reaction volume, carried out in a 96-well PCR reaction plate. The PCR reaction composition was: 4ul, 1×buffer II (Applied Biosystems, Foster City, CA), 1.5mM magnesium chloride, 0.4M betaine, 800μM dNTP mixture and 250nM of each primer. The mixture was kept at 95° C. for 10 minutes. The cycle reaction conditions were: 95° C. for 30 minutes, 48° C. for 30 minutes, and 72° C. for 30 min...
Embodiment 2
[0095] Example 2: High-throughput microbial identification method for extracts as proteins
[0096] (1) Sampling
[0097] Samples can come from various media such as throat swabs, blood, urine, pus, soil, and food. After melting or diluting the protective solution, the size of the sample is determined according to the volume of the adsorption column and the viscosity of the solution. .
[0098] (2) Extraction and purification of genetic material
[0099] The collection of the samples is performed due to the application of the principle of affinity chromatography. The adsorption column contains specific antibodies capable of adsorbing pathogenic proteins, and the type of antibodies can be changed according to the amount of the target. The chromatographic column after absorbing the sample is washed with washing solution to remove residual impurities.
[0100] (3) Enrichment
[0101] Then, the specific protein is eluted from the adsorption column by the eluent. After collect...
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Abstract
The invention discloses a high throughout microbiological assay method which comprises the following steps of sampling; extracting germ plasm; mass spectrometric analysis; data analysis; and microbiological assay. The method has the advantages that thousands of kinds of bacteria, virus, epiphytes and protozoans can be identified at the same time, while other systems only can identify little infective pathogene at the same time, so that the development of the systems are limited by quantity; the method can provide the relative quantity of a plurality of pathogens in a single sample, but most systems do not have the ability currently; the method is rapid and needs not to culture, one sample can be analyzed within less than 4 hours, while a plurality of weeks are spent on culturing, certainly, some samples can not be cultured at all; environmental or clinical samples which range from air to most complex biological samples in all mediums can be analyzed, so that the method is very universal; as no sequence measurement is needed, the method is the proposal with very good cost performance for the identification of infective pathogene.
Description
technical field [0001] The invention relates to a microorganism identification method, in particular to a high-throughput microorganism identification method with DNA, RNA or protein and other genetic materials based on PCR, protein purification and high-resolution tandem mass spectrometry technology. Background technique [0002] The traditional identification methods of microorganisms are based on the morphology, ecology, cell physiology, biochemistry and genes of microorganisms, and mainly include the following categories: biochemical methods, immunological techniques, molecular biology and molecular genetics methods, biological Sensors, chromatography technology. [0003] The above-mentioned methods all have the characteristics of being slow and unsuitable for large-scale screening and identification. They are only capable of identifying pathogenic microorganisms presumed to be present in the sample. [0004] Currently, there is no method capable of high-throughput ide...
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