A Rapid and Sensitive Reverse Transcription–Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for the Detection of Indian Citrus Ringspot Virus

Abstract

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the ‘Kinnow mandarin’, a commercial citrus crop cultivated in the north-west of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcriptase-polymerase chain reaction (RT-PCR) that is time-consuming. Here, we describe a novel, one-step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. The RT-LAMP assay was standardized by designing and testing four different primers that targeted the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. ICRSV-RT-LAMP assay developed in the present study is a simple, rapid, sensitive, and specific, technique. Moreover, the assay consists of only a single step and is more cost-effective than existing methods. This represents the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large scale indexing of field samples in diagnostic laboratories, nurseries, and for quarantine applications.

Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review

“…Various plant viruses like Barley yellow dwarf viruses [ 107 ], Cucumber mosaic virus [ 108 ], Potato leafroll virus [ 109 ], Southern tomato virus (STV) [ 110 ], Tomato brown rugose fruit virus (ToBRFV) [ 111 ], and Citrus leaf blotch virus in kiwifruit [ 112 ] have been detected using LAMP PCR and RT-PCR assays. Indian citrus ringspot virus (ICRSV) was analyzed using a novel, one-step RT-LAMP method by designing and testing four different primers that targets coat protein gene of ICRSV [ 113 ]. Warghane and colleagues [ 114 ] reported for the first time the development of RT-LAMP assays for rapid diagnosis of Indian Citrus tristeza virus (CTV) strains in citrus plant in India.…”

Fast, Precise, and Reliable Multiplex Detection of Potato Viruses by Loop-Mediated Isothermal Amplification

“…The detection of potato viruses typically requires field samples to be sent to a well-equipped laboratory for nucleic acid extraction and purification followed by RT-PCR and melt curve analysis. More recently, there has been an effort to replace the complex RT-PCR step with simpler isothermal amplification methods [38][39][40][41][42]. Therefore, we set out to develop a rapid, simple, and inexpensive approach for the detection of potato viruses that (a) minimizes sample processing; (b) provides reliable samples for the rapid and robust on-site diagnosis of infections by isothermal amplification; and (c) allows on-site verification by LFD analysis.…”

Exotic and Emergent Citrus Viruses Relevant to the Mediterranean Region

“…The sensitive and rapid detection of ICRSV was performed using the one-step RT-LAMP with four different primers, targeting the coat protein gene of the virus. The RT-LAMP assay was found to be rapid, simple, cost effective and about one hundred times more sensitive than RT-PCR [151].…”