TY - JOUR
T1 - High-resolution melting analysis
T2 - A novel approach for clade differentiation in Pythium insidiosum and pythiosis
AU - Worasilchai, Navaporn
AU - Permpalung, Nitipong
AU - Chindamporn, Ariya
N1 - Funding Information:
This work was supported by (1) the 90th Anniversary Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) year 2009 and (2) Chulalongkorn’s Research Repository ( CU-57-008-FW). We would like to extend our sincere gratitude toward Drs. Chakrabarti, Shivaprakash, and Ms. Sunita Gupta from Postgraduate Institute of Medical Education and Research, Chandigarh, India, for providing the fungal isolates of Zygomycetes group. Finally, we would like to sincerely thank all members in Mycology Unit, Department of Microbiology, King Chulalongkorn Memorial Hospital and Mycology research unit, Faculty of Medicine, Chulalongkorn University for all supports.
Publisher Copyright:
© The Author(s) 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.
PY - 2018/10/1
Y1 - 2018/10/1
N2 - Pythium insidiosum causes life-threatening human pythiosis. Based on phylogenetic analysis using internal transcribed spacer (ITS) region, mitochondrial cytochrome C oxidase II (COX2) gene, intergenic spacer (IGS) region and exo-1,3-β-glucanase gene (exo1), P. insidiosum is classified into clade A TH, B TH, and C TH related to geographic distribution. At present, polymerase chain reaction in any of these specific regions with DNA sequencing is the only technique to provide clade diagnosis. In this study, P. insidiosum-specific primers targeting COX2 gene were designed and used in real-time quantitative polymerase chain reaction (qPCR) with subsequent high-resolution melting (HRM) to provide rapid identification as well as clade classification for P. insidiosum. Based on the qPCR-HRM method, 15 P. insidiosum isolates could be differentiated from 28 related organisms with 100% specificity and 1 pg limit of detection. This technique was, in addition, directly tested on clinical samples from proved human pythiosis cases: nine corneal scrapes and six arterial clots. The qPCR-HRM results of all nine corneal samples were a 100% match with the results from the conventional PCR at clade level. However, the qPCR-HRM results of arterial clot samples were only matched with the nucleotide sequencing results from the conventional PCR at species level. In conclusion, the qPCR-HRM is a simple one closed tube, inexpensive and user-friendly method to identify P. insidiosum into clade level.
AB - Pythium insidiosum causes life-threatening human pythiosis. Based on phylogenetic analysis using internal transcribed spacer (ITS) region, mitochondrial cytochrome C oxidase II (COX2) gene, intergenic spacer (IGS) region and exo-1,3-β-glucanase gene (exo1), P. insidiosum is classified into clade A TH, B TH, and C TH related to geographic distribution. At present, polymerase chain reaction in any of these specific regions with DNA sequencing is the only technique to provide clade diagnosis. In this study, P. insidiosum-specific primers targeting COX2 gene were designed and used in real-time quantitative polymerase chain reaction (qPCR) with subsequent high-resolution melting (HRM) to provide rapid identification as well as clade classification for P. insidiosum. Based on the qPCR-HRM method, 15 P. insidiosum isolates could be differentiated from 28 related organisms with 100% specificity and 1 pg limit of detection. This technique was, in addition, directly tested on clinical samples from proved human pythiosis cases: nine corneal scrapes and six arterial clots. The qPCR-HRM results of all nine corneal samples were a 100% match with the results from the conventional PCR at clade level. However, the qPCR-HRM results of arterial clot samples were only matched with the nucleotide sequencing results from the conventional PCR at species level. In conclusion, the qPCR-HRM is a simple one closed tube, inexpensive and user-friendly method to identify P. insidiosum into clade level.
KW - Pythium insidiosum
KW - clade differentiation
KW - high-resolution melting
KW - pythiosis
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U2 - 10.1093/mmy/myx123
DO - 10.1093/mmy/myx123
M3 - Article
C2 - 29228389
AN - SCOPUS:85050919447
VL - 56
SP - 868
EP - 876
JO - Medical Mycology
JF - Medical Mycology
SN - 1369-3786
IS - 7
ER -