Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony.
In this work, loop-mediated isothermal amplification based detection assay using bacterial culture and bacterial colony for various common pathogens direct detection had been established, evaluated and further applied. A total of five species of common pathogens and nine detection targets (tlh, tdh and trh for V. Parahaemolyticus, rfbE, stx1 and stx2 for E. coli, oprI for P. aeruginosa, invA for Salmonella and hylA for L. monocytogenes) were performed on bacterial culture and bacterial colony LAMP. To evaluate and optimize this assay, a total of 116 standard strains were included. Then, for each detected targets, 20 random selected strains were applied. Results were determined through both visual observation of the changed color by naked eye and electrophoresis, which increased the accuracy of survey. The minimum adding quantity of each primer had been confirmed, and the optimal amplification was obtained under 65 °C for 45 min with 25 μl reaction volume. The detection limit of bacterial culture LAMP and PCR assay were determined to be 102and 104or 105CFU/reaction, respectively. No false positive amplification was observed when subjecting the bacterial -LAMP assay to 116 reference strains. This was the first report of colony-LAMP and culture-LAMP assay, which had been demonstrated to be a fast, reliable, cost-effective and simple method on detection of various common pathogens.
Department of Haematology, Guangzhou Women and Children's Medical Centre, Guangzhou Medical University, Guangzhou 510623, China. Electronic address: robinbobo@qq.com.
Muxia
M
Yan
PICU and Neonatology, Xiao Lan Hospital of Southern Medical University, Guangzhou 510180, China. Electronic address: 2821484106@qq.com.
Weidong
W
Li
Clinical Microbiology, Guangzhou Women and Children's Medical Centre, Guangzhou Medical University, Guangzhou 510180, China. Electronic address: 471122711@qq.com.
Zhenwen
Z
Zhou
Department of Haematology, Guangzhou Women and Children's Medical Centre, Guangzhou Medical University, Guangzhou 510623, China. Electronic address: chunpochui@qq.com.
Hongxia
H
Peng
Department of Haematology, Guangzhou Women and Children's Medical Centre, Guangzhou Medical University, Guangzhou 510623, China. Electronic address: 2261998630@qq.com.
Ziyan
Z
Luo
Department of Haematology, Guangzhou Women and Children's Medical Centre, Guangzhou Medical University, Guangzhou 510623, China. Electronic address: 574096897@qq.com.
Ling
L
Xu
D058491:Bacterial Load / Q000379:methods; D001431:Bacteriological Techniques; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
Bacterial-LAMP; Colony-LAMP; Common pathogens; Culture-LAMP; PCR