Simple and rapid bioluminescent detection of two verotoxin genes using allele‐specific PCR of E. coli O157: H7

Abstract

Allele-specific PCR for E. coli O157 was conducted with primers specific to verotoxin genes, verotoxin 1 (VT1) and verotoxin 2 (VT2). VT is an important cause of haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS) worldwide. We developed a simple, rapid bioluminescent detection method for E. coli O157. The method is based on the determination of pyrophosphoric acid (PPi) released during allele-specific PCR. Thus, released PPi is converted to ATP by ATP sulphurylase and the concentration of ATP is determined using the firefly luciferase reaction. As a result, VT1, VT2 and DNA with VT1/VT2 were clearly identified by this method. This protocol, which does not require expensive equipment, can be utilized to monitor the PCR product rapidly. Additionally, this methodology can be used as a high-throughput approach for measuring PCR products.

Rapid and highly sensitive detection of Escherichia coli O157:H7 in food with loop‐mediated isothermal amplification coupled to a new bioluminescent assay

“…Two distinct advantages of LAMP over PCR are that it amplifies at a constant temperature (≈60°C) and is tolerant of assay inhibitors, eliminating the need for a thermocycler or complicated sample preparation. Other attractive features of LAMP include its high specificity, high sensitivity, rapidity, and robustness [14]. LAMP is widely used in microbial identification, where it offers great advantages over PCR in terms of its rapid detection, high sensitivity, low cost, and simple operation.…”

Development of Highly Sensitive Analytical Methods for Biologically Relevant Materials and Their Pharmaceutical Applications

“…18) Although this method was applied to DNA analy- sis, the stability and sensitivity of the assay were inadequate due to the use of ATP sulfurylase. 19) We therefore developed a new pyrophosphate measurement method using pyruvate phosphate dikinase (PPDK) to replace ATP sulfurylase.…”

Detection of cariogenic bacteria genes by a combination of allele-specific polymerase chain reactions and a novel bioluminescent pyrophosphate assay

“…These bacteria were divided into three groups (Table 1): group 1, mixed sample of S. mutans and S. sobrinus; group 2, mixed sample of dex(+) bacteria (3-7) in Table 1; and, group 3, mixed sample of dex(À) bacteria (8)(9)(10)(11)(12)(13) in Table 1. DNA extracted from these groups was mixed and amplified by duplex PCR, which consisted of mutans PCR and sobrinus PCR, and by common PCR following bioluminescent pyrophosphate assay.…”

“…Previously, we developed a bioluminescent detection method for the O157 VT gene, which involved the 0003 luciferin-luciferase reaction following transformation of pyrophosphate produced during PCR to ATP by adenosine 5 0 -phosphosulfate (APS) 1 and ATP sulfurylase [8]. However, the sensitivity of this technique was insufficient due to the slight light emission of APS during the luciferin-luciferase reaction, leading to elevation of the blank value.…”