TY - JOUR
T1 - Development of a quantitative PCR assay for arcobacter spp. And its application to environmental water samples
AU - Shrestha, Rajani Ghaju
AU - Tanaka, Yasuhiro
AU - Malla, Bikash
AU - Tandukar, Sarmila
AU - Bhandari, Dinesh
AU - Inoue, Daisuke
AU - Sei, Kazunari
AU - Sherchand, Jeevan B.
AU - Haramoto, Eiji
N1 - Funding Information:
This study was supported by the Science and Technology Research Partnership for Sustainable Development (SATREPS) program of the Japan International Cooperation Agency (JICA) and the Japan Science and Technology Agency (JST), entitled “Hydro-microbiological Approach for Water Security in Kathmandu Valley,
Publisher Copyright:
© 2018, Japanese Society of Microbial Ecology. All rights reserved.
PY - 2018/9/29
Y1 - 2018/9/29
N2 - Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction–1 and sometimes detected as few as 3 copies reaction–1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3–9.1 log copies 100 mL–1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.
AB - Arcobacter spp. are emerging pathogens associated with gastroenteritis in humans. The objective of this study was to develop a highly sensitive and broadly reactive quantitative PCR (qPCR) assay for Arcobacter spp. and to apply the developed assay to different water sources in the Kathmandu Valley, Nepal. Fifteen samples to be analyzed by next-generation sequencing were collected from 13 shallow dug wells, a deep tube well, and a river in the Kathmandu Valley in August 2015. Among the 86 potential pathogenic bacterial genera identified, Acinetobacter, Pseudomonas, Flavobacterium, and Arcobacter were detected with relatively high abundance in 15, 14, 12, and 8 samples, respectively. A primer pair was designed with maximal nucleotide homologies among Arcobacter spp. by comparing the sequences of 16S rRNA genes. These primers were highly specific to most of the known species of Arcobacter and quantified between 1.0×101 and 6.4×106 copies reaction–1 and sometimes detected as few as 3 copies reaction–1. The qPCR assay was used to quantify Arcobacter spp. in bacterial DNA in not only the above 15 water samples, but also in 33 other samples collected from 15 shallow dug wells, 6 shallow tube wells, 5 stone spouts, 4 deep tube wells, and 3 springs. Thirteen (27%) out of 48 samples tested were positive for Arcobacter spp., with concentrations of 5.3–9.1 log copies 100 mL–1. This qPCR assay represents a powerful new tool to assess the prevalence of Arcobacter spp. in environmental water samples.
KW - Arcobacter spp
KW - Kathmandu Valley
KW - Next-generation sequencing
KW - Quantitative PCR
KW - Water source
UR - https://www.scopus.com/pages/publications/85054420707
U2 - 10.1264/jsme2.ME18052
DO - 10.1264/jsme2.ME18052
M3 - Article
C2 - 30185726
AN - SCOPUS:85054420707
SN - 1342-6311
VL - 33
SP - 309
EP - 316
JO - Microbes and Environments
JF - Microbes and Environments
IS - 3
ER -