Multiple internal controls enhance reliability for PCR and real time PCR detection of Rathayibacter toxicus

Mohammad Arif,Rachel Mann,Brendan Rodoni,James P Stack,Grethel Y Busot

doi:10.1038/s41598-021-87815-6

Abstract

Rathayibacter toxicus is a toxigenic bacterial plant pathogen indigenous to Australia and South Africa. A threat to livestock industries globally, the bacterium was designated a U.S. Select Agent. Biosecurity and phytosanitary concerns arise due to the international trade of seed and hay that harbor the bacterium. Accurate diagnostic protocols to support phytosanitary decisions, delineate areas of freedom, and to support research are required to address those concerns. Whole genomes of three genetic populations of R. toxicus were sequenced (Illumina MiSeq platforms), assembled and genomic regions unique to each population identified. Highly sensitive and specific TaqMan qPCR and multiplex endpoint PCR assays were developed for the detection and identification of R. toxicus to the population level of discrimination. Specificity was confirmed with appropriate inclusivity and exclusivity panels; no cross reactivity was observed. The endpoint multiplex PCR and TaqMan qPCR assays detected 10 fg and 1 fg of genomic DNA, respectively. To enhance reliability and increase confidence in results, three types of internal controls with no or one extra primer were developed and incorporated into each assay to detect both plant and artificial internal controls. Assays were validated by blind ring tests with multiple operators in three international laboratories.

Highlights

Rathayibacter toxicus is a gram-positive bacterial plant pathogen and a Select Agent in the United States since 2008 due to the potential threat to the U.S livestock industry
The genomic regions unique to three populations (RTI, RT-II and RT-III) of R. toxicus were identified by aligning the genomes from all three populations (Fig. 1)
We report the development of robust, population-specific and highly sensitive multiplex endpoint PCR and TaqMan qPCR assays for detection of the high consequence Select Agent, R. toxicus, but we describe the development and validation of a novel set of internal controls to enhance confidence in assay results

Summary

Introduction

Rathayibacter toxicus is a gram-positive bacterial plant pathogen and a Select Agent in the United States since 2008 due to the potential threat to the U.S livestock industry. TaqMan probe-based qPCR methods are used to detect target pathogen(s) with high sensitivity and reliability for applications in routine diagnostics, microbial forensics and plant b iosecurity[7,8,9]. Multiplex endpoint PCR methods are cost effective and easy to perform compared to TaqMan probe based multiplex qPCR m ethods[11]. For diagnostics requiring high confidence in test results, the inclusion of an internal control(s) in each PCR tube is extremely important to avoid the possibilities of false negative results. We developed accurate, reliable and sensitive diagnostic TaqMan probe-based multiplex real time qPCR and multiplex endpoint PCR for reliable detection and discrimination of the Select Agent, R. toxicus. The developed tools will provide capabilities for application in routine detection and discrimination of R. toxicus, ARGT disease outbreak management, agriculture biosecurity and R. toxicus population monitoring. Development of a customized artificial internal control may provide insight to develop AICs for other target pathogens

Methods

Results

Conclusion