One-step Multiplex Reverse Transcription-polymerase Chain Research Articles

A simplified, amplicon-based method for whole genome sequencing of human respiratory syncytial viruses

Human Respiratory Syncytial Virus (RSV) infections pose a significant risk to human health worldwide, especially for young children. Whole genome sequencing (WGS) provides a useful tool for global surveillance to better understand the evolution and epidemiology of RSV and provide essential information that may impact on antibody treatments, antiviral drug sensitivity and vaccine effectiveness. Here we report the development of a rapid and simplified amplicon-based one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) for WGS of both human RSV-A and RSV-B viruses. Two mRT-PCR reactions for each sample were designed to generate amplicons for RSV WGS. This new method was tested and evaluated by sequencing 206 RSV positive clinical samples collected in Australia in 2020 and 2021 with RSV Ct values between 10 and 32. In silico analysis and laboratory testing revealed that the primers used in the new method covered most of the currently circulating RSV-A and RSV-B. Amplicons generated were suitable for both Illumina and Oxford Nanopore Technologies (ONT) NGS platforms. A success rate of 83.5% with a full coverage for the genome of 98 RSV-A and 74 RSV-B was achieved from all clinical samples tested. This assay is simple to set up, robust, easily scalable in sample preparation and relatively inexpensive, and as such, provides a valuable addition to existing NGS RSV WGS methods.

Development of one-step multiplex RT-PCR assay for rapid simultaneous detection of five RNA viruses and Acidovorax citrulli in major cucurbitaceous crops in China.

Cucurbitaceous fruits and vegetables are important crops. Viral and bacterial diseases cause substantial economic losses to cucurbit crops globally. For rapid detection of these pathogens and improved disease control, a one-step multiplex reverse-transcription polymerase chain reaction (mRT-PCR) system was created. This method allowed for the concurrent detection of Tobacco mosaic virus (TMV), Zucchini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Cucumber green mottle mosaic virus (CGMMV), Cucumber mosaic virus (CMV), and Acidovorax citrulli. Five pairs of specific primers were created according to the conserved regions around the coat protein (CP) genes of each virus, and one pair was based on the A. citrulli internal transcribed spacer (ITS). To limit false negatives, one pair of primers, created based on the Transcriptional elongation factor 1-α (EF1-α) from the major cucurbitaceous crop species, was put into the mRT-PCR reaction system. Primer concentrations, annealing temperature, extension time, and amplification cycles were optimized. Anticipated fragments of 152bp (TMV), 205bp (ZYMV), 318bp (WMV), 419bp (CGMMV), 529bp (CMV), 662bp (A. citrulli), and 821bp (EF1-α) were amplified by the multiplex RT-PCR system, and their origin was established via DNA sequencing. This method was successfully used to examine field-collected seed samples of cucurbitaceous crops from China. The results demonstrated that the one-step mRT-PCR technique is a quick, efficient, and sensitive assay for the concurrent detection of six pathogens of cucurbits. It provides a method for monitoring and preventing these diseases.

A survey of viruses and viroids in astringent persimmon (Diospyros kakiThunb.) and the development of a one-step multiplex reverse transcription-polymerase chain reaction assay for the identification of pathogens

A survey of viruses and viroids in astringent persimmon (<i>Diospyros kaki</i>Thunb.) and the development of a one-step multiplex reverse transcription-polymerase chain reaction assay for the identification of pathogens

Differentiation of RotaTeq® vaccine strains from wild-type strains using NSP3 gene in reverse transcription polymerase chain reaction assay

RotaTeq® is a live attenuated human-bovine reassortant vaccine against rotaviruses that is used worldwide. However, shedding of the virus used in RotaTeq® has been detected in the feces of children following vaccination by the oral route, possibly affecting community immunity. Therefore, a simple and efficient method to discriminate between virulent and RotaTeq® vaccine strains is required. In this study, a novel one-step multiplex reverse-transcription polymerase chain reaction (RT-PCR) assay targeting the NSP3 gene was developed to detect RotaTeq® vaccine strains in fecal samples. RotaTeq® vaccine viruses were successfully distinguished from known wild-type rotavirus genotypes. In addition, the developed assay was able to detect rotaviruses in clinical stool samples obtained from South Korea during the 2011–2013 rotavirus seasons. Of the 1106 stool specimens from children with acute gastroenteritis that were screened, 286 rotaviruses were genotyped. RotaTeq® vaccine strains were identified in 39 samples (13.6%). The novel RT-PCR assay that was developed could be used to detect and discriminate between RotaTeq® vaccine strains that are shed in fecal matter, and to estimate the quantification of virus that has been shed after vaccination.

One-step multiplex reverse transcription-polymerase chain reaction for the simultaneous detection of three rice viruses

Rice stripe virus (RSV), Rice black-streaked dwarf virus (RBSDV), and Rice dwarf virus (RDV) are major rice-infecting viruses in Korea that can cause serious crop losses. A one-step multiplex reverse transcription-polymerase chain reaction (mRT-PCR) was developed for the simultaneous detection of these rice viruses. Three sets of specific primers targeted to the capsid protein coding genes of RSV, RBSDV, and RDV were used to amplify fragments that were 703bp, 485bp, and 252bp, respectively. The one-step mRT-PCR assay proved to be a sensitive and rapid method for detecting the three rice viruses. This method could be used to facilitate better control of rice viruses.

Multiplex reverse transcription PCR Luminex assay for detection and quantitation of viral agents of gastroenteritis

Background Several viruses can cause diarrheal disease, a leading cause of morbidity and mortality worldwide. Existing diagnostic methods include ELISA and nucleic acid amplification, usually performed individually. Objectives (1) To develop a multiplexed assay for simultaneous detection of major enteric viral pathogens. (2) Quantitation of viral load by normalizing with an extrinsic control. Study design A simple protocol combining a one-step multiplex reverse transcription-polymerase chain reaction (RT-PCR) with microsphere-based fluorescence detection was developed for norovirus GI and GII, rotavirus, astrovirus, sapovirus, and adenovirus. An extrinsic control, bacteriophage MS2, was spiked into each fecal sample before nucleic acid extraction to normalize between samples for the efficiency of nucleic acid extraction and amplification. Results The fluorescent results were quantitative and nearly as sensitive as the corresponding singleplex real time RT-PCR (qRT-PCR) assay on analytic samples. Upon testing 229 fecal samples from inpatients with diarrhea in Tanzania the assay yielded between 88% and 100% sensitivity and specificity for all analytes. The difference in fluorescence intensities of MS2 between samples indicated variable extraction efficiency and was used to better refine the viral load of each specimen. Conclusions This one-step nucleic acid-based assay enables rapid, sensitive and specific detection of the major viral causes of gastroenteritis. The quantitation yielded by the assay is informative for clinical research particularly in the context of mixed infections.

Improved multiplex reverse transcription-polymerase chain reaction to detect and identify five tospovirus species simultaneously

We improved a one-step multiplex reverse transcription-polymerase chain reaction (multi-PCR) method previously published for the simultaneous identification of five tospovirus species that mainly occur on Solanaceae and Compositae plants. The multi-PCR system is composed of a universal degenerate primer and five virus-species-specific primers that amplify bands unique to Iris yellow spot virus (837 bp), Tomato spotted wilt virus (709 bp), Impatiens necrotic spot virus (589 bp), Chrysanthemum stem necrosis virus (485 bp), and Capsicum chlorosis virus (366 bp). This technique is potentially useful for epidemiological studies of several tospoviruses that have recently been detected in previously uninfected fields of ornamental crops.