title: Development and application of a real-time polymerase chain reaction method for Campylobacter jejuni detection.
abstract: OBJECTIVE
  To develop a real-time polymerase chain reaction (PCR) method to detect and quantify Campylobacter jejuni (C. jejuni) from stool specimens.
  
  
  METHODS
  Primers and a probe for real-time PCR were designed based on the specific DNA sequence of the hipO gene in C. jejuni. The specificity of the primers and probe were tested against a set of Campylobacter spp. and other enteric pathogens. The optimal PCR conditions were determined by testing a series of conditions with standard a C. jejuni template. The detection limits were obtained using purified DNA from bacterial culture and extracted DNA from the stool specimen. Two hundred and forty-two specimens were analyzed for the presence of C. jejuni by direct bacterial culture and real-time PCR.
  
  
  RESULTS
  The optimal PCR system was determined using reference DNA templates, 1 × uracil-DNA glycosylase, 3.5 mmol/L MgCl2, 1.25 U platinum Taq polymerase, 0.4 mmol/L PCR nucleotide mix, 0.48 μmol/L of each primer, 0.2 μmol/L of probe and 2 μL of DNA template in a final volume of 25 μL. The PCR reaction was carried as follows: 95 °C for 4 min, followed by 45 cycles of 10 s at 95 °C and 30 s at 59 °C. The detection limit was 4.3 CFU/mL using purified DNA from bacterial culture and 10(3) CFU/g using DNA from stool specimens. Twenty (8.3%, 20/242) C. jejuni strains were isolated from bacterial culture, while 41 (16.9%, 41/242) samples were found to be positive by real-time PCR. DNA sequencing of the PCR product indicated the presence of C. jejuni in the specimen. One mixed infection of C. jejuni and Salmonella was detected in one specimen and the PCR test for this specimen was positive.
  
  
  CONCLUSIONS
  The sensitivity of detection of C. jejuni from stool specimens was much higher using this PCR assay than using the direct culture method.
authors.affiliation: State Key Laboratory for Infectious Disease Prevention and Control, and National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China. zhangmaojun@icdc.cn
authors.forename: Mao-Jun
authors.initials: MJ
authors.lastname: Zhang
authors.forename: Bo
authors.initials: B
authors.lastname: Qiao
authors.forename: Xue-Bin
authors.initials: XB
authors.lastname: Xu
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Zhang
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001431:Bacteriological Techniques; D002169:Campylobacter Infections / Q000175:diagnosis* / Q000382:microbiology; D016123:Campylobacter jejuni / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D002648:Child; D015169:Colony Count, Microbial; D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial / Q000032:analysis* / Q000302:isolation & purification; D005243:Feces / Q000382:microbiology*; D006801:Humans; D008875:Middle Aged; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction; D055815:Young Adult
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial
keywords: Application; Campylobacter jejuni; Real time polymerase chain reaction