A Multiplex Reverse Transcription (RT) High-Fidelity PCR Protocol for the Detection of Six Viruses that Cause Potato Tuber Necrosis

Abstract

Viruses that cause necrotic symptoms in potato tubers can be difficult to distinguish based on symptoms and frequently require multiple molecular tests to identify the pathogen. In this study, a multiplex RT PCR high fidelity PCR protocol was developed using previously validated primers that could accurately detect six important potato viruses and detect multiple viruses in a single sample simultaneously. To test the protocol, 53 tubers previously tested using conventional PCR were retested using this multiplex protocol and blindly evaluated. In every case, the multiplex PCR results were consistent with previous findings. In addition, the multiplex PCR protocol also detected several mixed infections that were previously undetected. The results demonstrate that this technique could be a valuable assay not only for diagnostic laboratories, but also for seed certification and regulatory agency laboratories to diagnose the virus(es) that may be present in tubers with surface or internal necrotic symptoms.Resumen Los virus que causan síntomas necróticos en tubérculos de papa pueden ser difíciles de distinguir con base en los síntomas, y frecuentemente se requiere de múltiples pruebas moleculares para identificar al patógeno. En este estudio, se desarrolló un protocolo múltiple de RT PCR de alta fidelidad de PCR utilizando iniciadores previamente validados que pudieran detectar con precisión seis virus importantes de papa y múltiples virus en una única muestra simultáneamente. Para probar el protocolo, se analizaron nuevamente 53 tubérculos que habían sido probados previamente usando la PCR convencional, utilizando este protocolo múltiple con evaluación ciega. En cada caso, los resultados de PCR múltiple fueron consistentes con lo que se encontró previamente. Además, el protocolo de PCR múltiple también detectó algunas mezclas de infecciones que previamente no se detectaron. Los resultados demuestran que esta técnica pudiera ser un ensayo valioso no solo para laboratorios de diagnóstico, sino también para certificación de semilla y para laboratorios de agencias de regulación para diagnosticar el (los) virus que pudieran estar presentes en tubérculos con síntomas necróticos superficiales o internos.

Viral metatranscriptomic approach to study the diversity of virus(es) associated with Common Bean (Phaseolus vulgaris L.) in the North-Western Himalayan region of India

“…Previous studies have shown the effectiveness of multiplex PCR in the simultaneous detection of three (Chiquito-Almanza et al, 2017 ), six (Cating et al, 2015 ), seven (Kwon et al, 2014 ; Zhao et al, 2015 ), eight (Kwak et al, 2014 ), and up to nine (Gambino, 2015 ) pathogens. The purpose of our optimization of multiplex PCR was to develop a robust, flexible, and accurate diagnostic tool for the detection of the identified viruses, as they produce similar symptoms and can be found within their hosts in multiple co-infections.…”

Silver-enhanced lateral flow immunoassay for highly-sensitive detection of potato leafroll virus

“…Compared to other techniques of PLRV detection, the proposed silver-enhanced LFIA is more sensitive than previously-described LFIAs in conventional format with an LOD of 30 ng/mL (Kondakova, Butenko, Skurat, & Drygin, 2016) and more rapid than tissue print immunoassay with a duration of 4-5 h (Samsatly, Jawhari, Najjar, Sobh, & Abou-Jawdah, 2014). Moreover, due to possibility of on-site application, this LFIA is a good alternative for PLRV assays based on reverse transcription polymerase chain reactions (Cating, Funke, Kaur, Hamm, & Frost, 2015;Ju, 2011;Zhang et al, 2017) or loop-mediated isothermal amplification (Ahmadi, Almasi, Fatehi, Struik, & Moradi, 2013;Almasi, Manesh, Jafary, & Dehabadi, 2013).…”

Current status and molecular phylogeny of some economically important viruses infecting wheat crops of Şırnak province, Turkey

“…Multiplex PCR assays were preferred by numerous plant virologists to define simultaneously multiple virus infections in cultivated crops (Nie and Singh, 2000;Saade et al, 2000). Concurrent amplification of plant-pathogen viruses, viroids, and phytoplasma by multiplex PCR assays was reported in sweet potato and potato (Kwak et al, 2014;Cating et al, 2015), in stone fruits (Sánchez-Navarro et al, 2005) in wheat (Tao et al, 2012;Hassan et al, 2018), in tobacco (Günay and Usta, 2020), in grapevine (Gambino and Gribaudo, 2006), in cucurbits (Kwon et al, 2014), in chrysanthemum (Zhao et al, 2015), in barley and triticale (Trzmiel, 2017), in the citrus tree (Roy et al, 2005), and in garlic (Park et al, 2005).…”