title: Single-tube nested PCR assay for the detection of avian botulism in cecal contents of chickens.
abstract: This paper describes a novel diagnostic method for the detection of avian botulism caused by Clostridium botulinum type C and C/D, using single-tube nested PCR assay. This assay was developed to overcome the disadvantages of bioassays used in experiments with mice. Three primer pairs including an antisense primer were designed to target the N-terminal of the toxin gene from C. botulinum types C and C/D. The specificity of the PCR assay was confirmed by using 33 bacterial strains and chicken cecal contents from farms that experienced botulism outbreaks. The detection limit for purified DNA was 1.1 fg/μl, and for bacterial spores was 4.3 spores/200 mg of cecal contents. While checking for specificity of the PCR assay, the reactions with the templates form C. botulinum type C and C/D which were tested became positive, but the rest of the reactions turned negative. However, the results for all clinical samples (n = 8) were positive. The PCR assay results for cecal samples obtained from 300 healthy chickens (150 Korean native chickens and 150 broilers) were all negative. This assay is rapid and straightforward and evades ethical issues associated with mouse bioassay. Moreover, it is more economical than real-time PCR.
authors.affiliation: Avian Disease Division, Animal and Plant Quarantine Agency, 335 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
authors.forename: Il
authors.initials: I
authors.lastname: Jang
authors.affiliation: College of Veterinary Medicine, Chonnam National University, 300 Yonbongdong, Buk-gu, Gwangju 500-757, Republic of Korea.
authors.forename: Jae-Il
authors.initials: JI
authors.lastname: Lee
authors.affiliation: General Service Division, Animal and Plant Quarantine Agency, 335 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
authors.forename: Yong-Kuk
authors.initials: YK
authors.lastname: Kwon
authors.affiliation: Avian Disease Division, Animal and Plant Quarantine Agency, 335 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
authors.forename: Min-Su
authors.initials: MS
authors.lastname: Kang
authors.affiliation: Avian Disease Division, Animal and Plant Quarantine Agency, 335 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
authors.forename: Hye-Ryoung
authors.initials: HR
authors.lastname: Kim
authors.affiliation: Avian Disease Division, Animal and Plant Quarantine Agency, 335 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
authors.forename: Ji-Young
authors.initials: JY
authors.lastname: Park
authors.affiliation: Avian Disease Division, Animal and Plant Quarantine Agency, 335 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
authors.forename: Song-Hyun
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Avian Disease Division, Animal and Plant Quarantine Agency, 335 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea.
authors.forename: Hee-Soo
authors.initials: HS
authors.lastname: Lee
authors.affiliation: Avian Disease Division, Animal and Plant Quarantine Agency, 335 175 Anyang-ro, Manan-gu, Anyang-si, Gyeonggi-do 430-757, Republic of Korea. Electronic address: kyusfather@korea.kr.
authors.forename: You-Chan
authors.initials: YC
authors.lastname: Bae
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques / Q000379:methods*; D001905:Botulinum Toxins / Q000235:genetics; D001906:Botulism / Q000175:diagnosis / Q000662:veterinary*; D002645:Chickens; D003014:Clostridium botulinum / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011201:Poultry Diseases / Q000175:diagnosis* / Q000382:microbiology; D012680:Sensitivity and Specificity; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D017931:DNA Primers; C096325:botulinum toxin type D; D001905:Botulinum Toxins; C096324:botulinum toxin type C
keywords: Avian botulism; Clostridium botulinum; Single-tube nested PCR