Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus Y

Mohammad Amin Almasi

doi:10.4172/2157-7471.1000188

Abstract

Loop-Mediated Isothermal Amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. To diminish the time required for some diagnostic assays including Reverse Transcription PCR (RT-PCR), Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) and also DAS-ELISA into a minimum level, an innovative colorimetric IC-RT-LAMP and IC-RT-PCR protocol on the basis of Potato Virus Y (PVY) genome were used and optimized. Firstly, DAS-ELISA assay was performed to detect of the virus in a collection containing 95 suspicious samples. Lastly, five samples were detected as the positive samples. Then, the positive samples were verified by molecular methods. In this regard, all four RT-LAMP primers (i.e. F3, B3, FIP and BIP) together with RT-PCR primers (F and B) were selected on the basis of coat protein gene (CP) of PVY genome. Even though DAS-ELISA, RT-PCR and RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Furthermore, the results demonstrated that the RT-LAMP assay was 100 times sensitive and 3 time faster compared to RT-PCR. LAMP assay was accomplished in the water bath either frees from any thermal cycler machine or sophisticated laboratories facility. Meanwhile, among six different visual dyes to accurately detect IC–RT-LAMP products, Hydroxynaphthol blue, GeneFinderTM and SYBR Green I could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. We accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PVY recognition and probably other viral-based diseases.

Highlights

Potato (Solanum tuberosum L.), as the world’s most important nongrain food crop [1]
A large number of studies have been accomplished using loop-mediated isothermal amplification (LAMP) or RTLAMP including Potato virus Y [4], Potato Leafroll virus [18], Japanese yam mosaic potyvirus [19], Tomato Yellow Leaf Curl virus [20], Rabies virus [21], Macrobrachium rosenbergii nodavirus [22] and Cymbidium mosaic virus [23], all of which basically require more precise extraction protocol(s) about generating higher concentrations of DNA or RNA followed by the least amounts of probable contaminations which act as inhibitors in an amplification process
A fragment with the size band of 480 bp was detected when the Reverse Transcription Polymerase Chain Reaction (PCR) (RT-PCR) products were run on 1.5% agarose gel and stained with ethidium bromide (Figure 1a)

Summary

Introduction

Potato (Solanum tuberosum L.), as the world’s most important nongrain food crop [1]. Potato plants, like other crops, are exposed frequently to plenty of infectious viruses, economically the most important of which are Potato leafroll virus (PLRV, genus Polerovirus), Potato virus Y (PVY, genus Potyvirus), Potato virus X (PVX, genus Potexvirus) and Potato virus S (PVS, genus Carlavirus) [2]. A large number of studies have been accomplished using LAMP or RTLAMP including Potato virus Y [4], Potato Leafroll virus [18], Japanese yam mosaic potyvirus [19], Tomato Yellow Leaf Curl virus [20], Rabies virus [21], Macrobrachium rosenbergii nodavirus [22] and Cymbidium mosaic virus [23], all of which basically require more precise extraction protocol(s) about generating higher concentrations of DNA or RNA followed by the least amounts of probable contaminations which act as inhibitors in an amplification process. Despite a few number of studies about immunocapture reverse transcription loopmediated isothermal amplification (IC–RT-LAMP) [17,24] because the technique has not been yet introduced for detection of PVY, an attempt was made to optimize a new protocol of it to save time and remove RNA extraction. Since the existence of one-step IC-RT-LAMP positive amplicons has been proved to be confirmed by adding a number of fluorescent dsDNA intercalating dye including ethidum bromide [25], SYBR Green [26] and propidium iodide [27] after the reaction is completed or metal indicators such as calcein [28], Gene-FinderTM [29,30], hydroxyl naphthol blue [31,32,33] and magnesium pyrophosphate [17,18] prior to the reaction, allowing observation with the naked eye, here, six different visualization systems were employed to assess the colour stability as well as safety feature of each one in a viral detection procedure of PVY

Methods

Results

Discussion

Conclusion