Real time PCR using TaqMan and SYBR Green for detection of Enterobacter sakazakii in infant formula.
Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, we developed two real time PCR assays using TaqMan and SYBR Green to identify the pathogen after selective enrichment in mLST and BHI. The accuracy of two detections was tested by 35 strains of E. sakazakii and 88 non-E. sakazakii bacterial strains. The results showed that all of these E. sakazakii strains were positive reaction to the detections and all of the non-E. sakazakii strains were negative. The newly developed assays enable us to detect 1.1 CFU/100 g infant formula. And both of the assays can be accomplished within 2 business days. Compared to the traditional detection, the real time PCR procedures are quicker and simpler. In this study, we also developed a new method to design the primers, which can support multiple real time PCR with one pair of primers in SYBR Green detection. The detection methods are more sensitive and effective based on Two-Tm-Value of PCR.
Liu
Yin
Y
Department of Biochemistry and Molecular Biology, College of Life Science, Nankai University, Tianjin 300071, China.
Cai
Xiaoning
X
Zhang
Xia
X
Gao
Qili
Q
Yang
Xiaochuan
X
Zheng
Zejun
Z
Luo
Maohuang
M
Huang
Xitai
X
D000818:Animals; D001483:Base Sequence; D052160:Benzothiazoles; D002417:Cattle; D044083:Cronobacter sakazakii / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000737:chemistry / Q000235:genetics; D003959:Diamines; D005516:Food Microbiology; D041943:Infant Formula; D008892:Milk / Q000382:microbiology*; D008969:Molecular Sequence Data; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D011804:Quinolines; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase / Q000737:chemistry
D052160:Benzothiazoles; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012336:RNA, Ribosomal, 16S; D012338:RNA, Ribosomal, 23S; C098022:SYBR Green I; D019914:Taq Polymerase