美国食品药品局细菌分析手册（BAM）第28章：产肠毒素霍乱弧菌的检测（2001年1月版）BAM Chapter 28 Detection of Enterotoxigenic Vibrio cholerae

Recent epidemics of cholera in various parts of the world have emphasized the urgent need for rapid and reliable detection methods for Vibrio cholerae, especially in food and water. Classical microbiological methods are sensitive and specific; however, they require several days to complete (see Chapter 9) and may result in considerable loss of perishable foods. Since cholera toxin production (encoded by the ctxAB genes) is the major factor in the pathogenesis of cholera, a polymerase chain reaction (PCR) method that selectively amplifies a DNA fragment within the ctxAB operon of V. cholerae has been developed and applied to various foods (19).
The PCR was first described by Mullis et al. in 1985, and since then has revolutionized most of the biological sciences (25). In this technique, double-stranded target DNA is denatured to provide single-stranded templates to which specific oligonucleotide primers are hybridized, followed by primer extension with a thermostable DNA polymerase (26). Primer pairs complementary to opposite strands of a DNA region are chosen so that 5' to 3' directional extensions are toward one another. Thus repetitive denaturation, annealing, and primer extension cycles exponentially amplify a unique DNA fragment bordered by the primers. The process is extremely rapid (as little as 30 min for 25 cycles with certain thermocyclers) and sensitive (amplification of gene sequences from a single cell is possible). Further, the process can be designed to be specific for genus, species, or allele.
Use of the PCR as a detection method for microbial pathogens in foods has been documented in approximately two dozen PCR-based, detection procedures published by early 1994 (Table 1). PCR-based methods have been developed to detect a wide variety of foodborne pathogens, including Listeria monocytogenes, enterotoxigenic Escherichia coli (see Chapter 4), V. vulnificus, V. cholerae, Shigella flexneri, Yersinia enterocolitica, various Salmonella and Campylobacter species, and the Hepatitis A (15) (see Chapter 26) and Norwalk viruses.

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