A comparison of ZEN double-quenched probe and SYBR GreenER chemistries in the real-time PCR based quantitative detection of enterotoxigenic Bacillus cereus in milk
- Author:
Nur Thaqifah SALIHAH
1
;
Mohammad Mosharraf HOSSAIN
;
Mas Rina Wati Abdul Hamid
;
Minhaz Uddin AHMED
Author Information
1. Biosensors and Biotechnology Laboratory, Integrated Science Building, Faculty of Science, Universiti Brunei Darussalam, Jalan Tungku Link, Gadong BE 1410, Brunei Darussalam
- Publication Type:Journal Article
- Keywords:
Real-time PCR;
Bacillus cereus;
DNA;
Bacteria;
ZEN™ double-quenched probe;
SYBR GreenER™
- From:Malaysian Journal of Microbiology
2018;14(1):34-40
- CountryMalaysia
- Language:English
-
Abstract:
Aims: Comparison between ZEN™ double-quenched probe and SYBR GreenER™ real-time PCR assay to develop asensitive and specific assay for the direct detection and quantification of enterotoxigenic Bacillus cereus in milk.Methodology and results: Novel primers and probe were designed to target the enterotoxigenic nhe gene. Theperformance of ZEN™ double-quenched probe and SYBR GreenER™ chemistry were compared by using knownconcentrations of purified DNA. ZEN™ double-quenched probe showed a dynamic range of 3 log units and sensitivity of600 fg/reaction or 100 copies/reaction. SYBR GreenER™ chemistry had a wider quantitative dynamic range of 6 logunits with sensitivity down to 6 fg/reaction or 1 copy number/reaction. Thus, SYBR GreenER™ chemistry was 100×more sensitive with wider quantification range compared to ZEN™ probe chemistry. Similar result was also found forSYBR GreenER™ assay and ZEN™ probe chemistry in DNA extracted directly from artificially inoculated milk, with thelowest limit of detection by SYBR GreenER™ assay in the range of 6 fg/reaction or 25 copies/mL and it quantifiedBacillus cereus in milk with high relative accuracy.Conclusion: SYBR GreenER™ assay provides a fast, sensitive and specific detection and quantification ofenterotoxigenic Bacillus cereus and allowed a direct assessment and quantification of Bacillus cereus from milk foodsample.Conclusion, significance and impact of study: The study shows an efficient, specific and highly sensitive method ofdirectly assessing the enterotoxigenic Bacillus cereus from milk product, using cheaper dsDNA binding SYBRGreenER™ dye.
- Full text:20.2018my0420.pdf
