PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer.
A PCR detection based on 16S-23S rDNA internal transcribed spacer (ITS) of Klebsiella pneumoniae was developed in the present study. Nineteen different ITS sequences were amplified from 6 strains of K. pneumoniae by universal primers. By sequencing and alignment of these sequences to the other homologous in GenBank, species-specific primers of K. pneumoniae, Pf/Pr1 and Pf/Pr2, were designed for amplification of the ITS sequence from the operon containing tDNA(Ala) and tDNA(Ile). Ten type strains and 21 isolates of K. pneumoniae were positive to the PCR detection, and all of the non-K. pneumoniae reference strains (79 strains) were negative. The enrichment was performed in this procedure with a modified growth media to enrich K. pneumoniae from 1.5 CFU/100 g infant formula to about 10(5) CFU/ml in 900 ml of the media. Combination of the enrichment, with the PCR assay can detect 1.5 CFU/100 g infant formula of K. pneumoniae within 48 h. Furthermore, K. pneumoniae strains KPE050803 and KPE 050830 were identified by this method in 63 infant formula samples.
Liu
Yin
Y
Department of Biochemistry and Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300457, China.
Liu
Chao
C
Zheng
Wenjie
W
Zhang
Xia
X
Yu
Jia
J
Gao
Qili
Q
Hou
Yanmei
Y
Huang
Xitai
X
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D021901:DNA, Intergenic / Q000235:genetics*; D021903:DNA, Ribosomal Spacer; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D006801:Humans; D007223:Infant; D007225:Infant Food / Q000382:microbiology*; D041943:Infant Formula; D007231:Infant, Newborn; D007711:Klebsiella pneumoniae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000235:genetics; D021521:Ribotyping; D017421:Sequence Analysis; D013045:Species Specificity
D004269:DNA, Bacterial; D021901:DNA, Intergenic; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S