Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4.

Ying-Hong Lin,Tzu-Yu Chen,Yi-Jia Lin,Li-Ling Hong,Tsai-De Chang,Pi-Fang Linda Chang,Thumballi R Ganapathi

doi:10.1371/journal.pone.0159681

Ying-Hong Lin, Tzu-Yu Chen + Show 5 more

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https://doi.org/10.1371/journal.pone.0159681

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Abstract

This study developed a novel and inexpensive detection method based on a TaqMan probe-based insulated isothermal polymerase chain reaction (iiPCR) method for the rapid detection of Panama disease caused by Fusarium oxysporum f. sp. cubense (Foc) race 4, which is currently among the most serious fungal vascular diseases worldwide. By using the portable POCKIT™ device with the novel primer set iiFoc-1/iiFoc-2, the Foc race 4 iiPCR assay (including DNA amplification and signal monitoring) could be completed within one hour. The developed Foc race 4 iiPCR assay is thus a user-friendly and efficient platform designed specifically for the detection of Foc race 4. The detection limit of this optimized Foc iiPCR system was estimated to be 1 copy of the target standard DNA as well as 1 fg of the Foc genomic DNA. This approach can serve as a rapid detection method for in planta detection of Foc race 4 in field-infected banana. It was concluded that this molecular detection procedure based on iiPCR has good potential for use as an efficient detection method.

Highlights

Bananas and plantains, members of the Musa genus, are cultivated in humid tropical areas worldwide, making them one of the most economically important and popular fruits crops in the world
The soil-borne fungal disease, banana Fusarium wilt (FW), commonly known as Panama disease, which is caused by Fusarium oxysporum f. sp. cubense (Foc), is a highly-lethal vascular fungal disease in banana plants
In order to develop a realizable insulated isothermal polymerase chain reaction (PCR) (iiPCR) detection system for Foc race 4, the novel iiPCR primer set iiFoc-1/iiFoc-2 and TaqMan probe iipFoc-1 were developed according to the sequence of the Foc race 4-specific marker Foc242

Summary

Introduction

Members of the Musa genus, are cultivated in humid tropical areas worldwide, making them one of the most economically important and popular fruits crops in the world. The soil-borne fungal disease, banana Fusarium wilt (FW), commonly known as Panama disease, which is caused by Fusarium oxysporum f. Cubense (Foc), is a highly-lethal vascular fungal disease in banana plants. When Foc attacks a banana plant, it leads to yellowing and wilting of the older leaves that progresses to the younger leaves until the whole plant is killed. This lethal effect makes Panama disease the major limiting factor for banana production worldwide [1].

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