Background[1-3]
Phosphate glucose peptone water medium can be used for methyl red and V-P tests of bacteria. Polypeptone provides carbon and nitrogen sources, vitamins and growth factors; glucose is fermentable sugar; dipotassium hydrogen phosphate is a buffer; sodium chloride maintains balanced osmotic pressure.
Methyl red (MR) test: Enterobacteriaceae bacteria decompose glucose during sugar metabolism to produce pyruvate, which is then decomposed to produce formic acid, acetic acid, lactic acid, and succinic acid. When E. coli decomposes pyruvate, more secondary acids are produced, the pH is 4.5 or lower, and methyl red appears red (positive). In the culture solution of Aerobacillus aerogenes, part of the pyruvate turns into neutral acetyl methyl carbinol, less acids are generated, the pH is above 5.4, and methyl red appears orange (negative).
Acetyl methyl methanol production test (V-P test): Bacteria ferment glucose to generate pyruvate, and some bacteria can decarboxylate pyruvate to form acetyl methyl methanol. In alkaline solution, acetyl methyl carbinol can be oxidized to diacetyl (butanedione) in air. Diacetyl then reacts with the guanidine group of arginine in peptone to produce a red compound. α-Naphthol was added as a catalyst in the test to deepen the color of the reaction and improve the sensitivity of the reaction.
Application[4][5]Moistening soil
Research on toxin genes used to detect pathogenic E. coli in surface waters of the Minjiang River Basin using M-PCR
Using Escherichia coli isolated from the surface water of the Minjiang River Basin as material, fecal Escherichia coli was isolated through membrane filtration method combined with mFC-BCIG culture medium, and lactose fermentation culture medium and phosphoric acid were used Salt glucose peptone water culture medium was used to perform biochemical testing on it, and finally the number of fecal E. coli in unit volume of water sample was determined, and then the fecal E. coli strain was made into a PCR template and stored in glycerol.
The research results show that: Escherichia coli was isolated and identified and multiplex PCR detection of 12 toxin genes was conducted in 7 sections from the upper reaches to the lower reaches of the Minjiang River Basin. Ten toxin genes were detected in 1231 strains of fecal E. coli identified, among which E. coli with diffusible adhesin (aidA-1) toxin gene accounted for 12.99% of the total, heat-labile enterotoxin (elt) and heat-stable enterotoxin (elt). The number of toxins (astA) was second (accounting for 5.8% and 5.4% of the total, respectively), and the main pathogenic factors of a few high-risk strains were also detected, such as Shigella toxin (Stx2e) and drug resistance factor (sepA). Water samples were taken from the Hongshan Bridge and Jiejie Bridge sections in the Fuzhou crossing section of the Minjiang River Basin in different seasons to isolate and identify E. coli.
Qualitative and quantitative analysis of the spatiotemporal distribution of Escherichia coli toxin genes isolated in different seasons was performed using multiplex PCR method. From the 2015 strains of fecal E. coli isolated, 840 potentially pathogenic E. coli strains were found, belonging to enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), enteropathogenic E. coli (EPEC) and Enterotoxigenic Escherichia coli (ETEC).
Among the 10 toxin genes detected, diffusible adhesin gene (aidA-1), heat-stable enterotoxin gene (astA) and drug resistance factor (sepA) were the most common; total bacterial count, E. coli The total number and total number of pathogenic E. coli are lower in winter, more in spring, and reach the peak of the whole year in summer and autumn. The types and numbers of toxin genes of potential pathogenic E. coli are also the least in winter, increase in spring and summer, and reach the peak in autumn. most.
Use multiplex PCR to detect the virulence genes in fecal E. coli strains, providing scientific basis and technical support for the pollution status and risk assessment of the Minjiang River.
