Typing of Clostridium perfringens by in vitro amplification of toxin genes

Abstract

The strains of Clostridium perfringens are classified according to major toxins produced. Classically, this determination involves the seroneutralization of their lethal effect in mice. However, this method requires specific antisera and a large number of mice. In this work, a new typing method was developed based on the amplification of toxin genes by polymerase chain reaction (PCR). By combination of several pairs of primers, the toxinotype of a Cl. perfringens strain was determined by looking at the pattern of bands on an agarose gel electrophoresis. This mixture contained primers amplifying simultaneously a part of alpha-toxin, beta-toxin, epsilon-toxin and enterotoxin genes. In order to distinguish between toxinotype A and E, the l-toxin gene fragment must be amplified in a separate PCR reaction. Moreover, with the primers combination, in most cases, a PCR product corresponding to the alpha-toxin gene was obtained from direct enrichments of animal intestinal contents.

PCR detection of Clostridium perfringens producing different toxins in faeces of goats

“…The tests of Daube et al (1994) and Yoo et al (1997) employed The PCR products amplified in this study (Fig. 1) are in extracted and purified DNA from target organisms, while the agreement with previous reports by Daube et al (1994) and present method employs bacterial biomass in a direct lysis Havard et al (1992), who used the same primer sets and PCR procedure. This is the most likely explanation for their obtained similar PCR products using Cl.…”

“…Although the present method requires the preparation, Daube et al (1994) reported a method for toxin typing of amplification and electrophoresis of four separate PCRs per pure cultures of Cl. perfringens in a multiplex PCR using the sample, it does avoid the time-consuming and costly process same alpha, beta and iota primer sets as used here, while their of DNA extraction and purification.…”

“…Hence, the present epsilon primers were different to the present study. Although authors think their method is a useful alternative to the Daube et al (1994) were able to amplify alpha, beta and multiplex PCRs based on purified DNA, particularly in diagnostic laboratories where the cost and speed are essential …”

“…In the test by Daube et al (1994), Cl. perfringens types B and D showed a band at the 206 bp level for the epsilon toxin.…”

Detection of Clostridium septicum Hemolysin Gene by Polymerase Chain Reaction.

“…By this technique, organisms can be detected and identified directly from clinical specimens or environmental samples. Recently, PCR has been used for the detection of toxin genes, including neurotoxins of C. botulinum type A to F [4][5][6][7][18][19][20], major toxins and enterotoxin of C. perfringens [3,10,16], and toxin A of C. difficile [8,[13][14].…”