Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) assays for the detection of two novel viruses infecting ginger

Highlights

• RT-LAMP and RT-RPA based isothermal assays were developed for quick detection of two novel viruses infecting ginger.

• Detection limits of viruses in plants by RT-LAMP and RT-RPA was determined and compared with conventional and real-time RT-CR.

• RT-LAMP was 1000 times more sensitive than conventional RT-PCR while it was 100 times less sensitive than real-time RT-PCR.

• RT-RPA was 100 to 1000 times more sensitive than conventional RT-PCR while it was 100 to 1000 times less sensitive than real-time RT-PCR.

• Both RT-LAMP and RT-RPA assays were validated by testing field samples of ginger collected from different regions.

Abstract

Our recent studies have shown the association of two novel viruses namely, ginger chlorotic fleck-associated virus 1 (GCFaV-1) and ginger chlorotic fleck-associated virus 2 (GCFaV-2) with chlorotic fleck disease of ginger. As ginger is propagated through vegetative means, the development of diagnostics would aid in the identification of virus-free plants. In the present study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) and reverse transcription recombinase polymerase amplification (RT-RPA) assays were developed and validated for the quick detection of GCFaV-1 and GCFaV-2. The detection limits of viruses by these assays, when compared with conventional and real-time RT-PCR, showed that RT-LAMP was up to 1000 times more sensitive than conventional RT-PCR and one-hundredth that of real-time RT-PCR for both the viruses. The detection limit of RT-RPA for GCFaV-1 was up to 100 times more than that of RT-PCR and one-thousandth that of real-time RT-PCR. On the other hand, for detecting GCFaV-2, RT-RPA was found up to 1000 times more sensitive than conventional RT-PCR and one hundredth that of real-time RT-PCR. Based on the cost-effectiveness and duration, RT-LAMP and RT-RPA assays can be suggested for the rapid detection of both viruses.

Introduction

Ginger (Zingiber officinale Rosc.) is an important herbaceous perennial that belongs to the family Zingiberaceae, grown in different parts of the world. Ginger is widely used as a spice and herbal medicine all around the globe due to its high nutritive and medicinal properties (Dhanik et al., 2017). Even though fungal and bacterial diseases are the most devastating, viral diseases are also important to ginger plants considering the vegetative propagating nature of ginger and transmission of viruses. The viral disease was first reported by Nambiar and Sharma (1974) in both exotic and indigenous types of ginger. Later, Thomas (1986) described an isometric ssRNA virus with a possible affinity to the sobemo-virus group infecting ginger with chlorotic fleck symptoms. However, the exact causal virus associated with the disease remained unidentified until recently. Our recent study based on the small RNA and transcriptome sequencing of chlorotic fleck infected ginger for the first time showed the presence of two new viruses. Of these, cloning and sequencing of full genome of the isometric virus were recognized as a novel member of the family Tombusviridae (GenBank accession number MN581046) while partial genome analysis of the flexuous rod-shaped virus was recognized as a novel member of the genus, Ampelovirus (GenBank accession number MN581045) (Bhat et al., 2020, unpublished). The tentative names corresponding to these viruses are ginger chlorotic fleck-associated virus 1 (GCFaV-1) and ginger chlorotic fleck-associated virus 2 (GCFaV-2).

Identification of virus-free plants and their propagation is necessary to produce virus-free planting materials. Serological methods like ELISA and molecular methods like PCR, qPCR are common ways of detecting viruses in plants. Loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) are other two reliable and sensitive isothermal molecular assays used to detect viruses (Peng et al., 2012; Mekuria et al., 2014). Unlike PCR based techniques, these methods are less time consuming, relatively inexpensive, and can be carried out at a single constant temperature. LAMP includes six specially designed primers matching the target DNA fragment and Bst polymerase, and the DNA is amplified by strand-displacing activity under isothermal conditions (60–65 ℃) within a short time (1 h). Reverse transcription loop-mediated isothermal amplification (RT-LAMP) method has been reported for the detection of RNA viruses successfully and efficiently with reverse transcriptase addition to the LAMP components (Nie, 2005; Le et al., 2010; Bhat et al., 2013). LAMP and RT-LAMP products can be viewed either by colour changes by adding certain dyes in the reaction mix or as a ladder-like pattern on gel electrophoresis (Tomita et al., 2008). LAMP and RT-LAMP techniques were employed for detection of various DNA and RNA viruses such as Japanese yam mosaic virus (Fukuta et al., 2003), rice grassy stunt virus, rice tungro spherical virus, and rice tungro bacilliform virus (Le et al., 2010), banana bunchy top virus (Peng et al., 2012), piper yellow mottle virus, cucumber mosaic virus (Bhat et al., 2013), rice ragged stunt virus (Lai et al., 2018) and turnip yellows virus (Congdon et al., 2019).

Recombinase polymerase amplification (RPA) is another isothermal amplification assay for nucleic acid detection (Piepenburg et al., 2006). RPA uses an enzymatic mixture that contains recombinase, single-strand DNA binding proteins, and a DNA polymerase with strand-displacement activity for amplification of the target sequence which can be visualized in gel electrophoresis. Amplification products of RPA can be readily cloned and sequenced directly, as PCR products, but unlike LAMP where multimeric products form. Reverse transcription (RT) - RPA can be successfully carried out with synthesized cDNA from isolated RNA. RPA was used for the detection of viruses with DNA genomes such as bean golden yellow mosaic virus, tomato mottle virus and tomato yellow leaf curl virus (Londoño et al., 2016), banana bunchy top virus (Kapoor et al., 2017) and piper yellow mottle virus (Mohandas and Bhat, 2020). RT-RPA was used for the detection of viruses with RNA genomes such as yam mosaic virus (Silva et al., 2015), rose rosette virus (Babu et al., 2017), maize chlorotic mottle virus (Jiao et al., 2019) and cucumber green mottle mosaic virus (Zeng et al., 2019). The present study describes the development of RT-LAMP and RT-RPA assays for the detection of GCFaV-1 and GCFaV-2 associated with chlorotic fleck disease in ginger and comparison of their sensitivity with RT-PCR and real-time RT-PCR.