scholarly journals Developing a One-Step Multiplex PCR Assay for Rapid Detection of Four Stubby-Root Nematode Species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus

Plant Disease ◽  
2019 ◽  
Vol 103 (3) ◽  
pp. 404-410 ◽  
Author(s):  
Danqiong Huang ◽  
Guiping Yan ◽  
Neil Gudmestad ◽  
Weimin Ye ◽  
Jonathan Whitworth ◽  
...  
Keyword(s):  
United States ◽  
Multiplex Pcr ◽  
The United States ◽  
Parasitic Nematodes ◽  
Diagnostic Efficiency ◽  
Pcr Assay ◽  
Its1 Region ◽  
Multiplex Pcr Assay ◽  
One Step ◽  
Plant Parasitic

Four trichodorid species, Paratrichodorus allius, P. minor, P. porosus, and Trichodorus obtusus, were found in multiple states in the United States. Traditional diagnosis based on morphology and morphometrics is laborious and requires an experienced taxonomist. Additionally, end-point diagnosis using PCR was only available for P. allius. To increase diagnostic efficiency and reduce costs, a one-step multiplex PCR assay was developed to simultaneously identify these four species using one PCR reaction. Available sequences of 18S ribosomal DNA and internal transcribed spacer 1 (ITS1) region of these species were aligned and five primers were designed. The conserved forward primer located in the 18S region, in combination with the species-specific antisense primer in the ITS1 region, amplified a single distinctive PCR fragment for each species (421/425 bp for P. allius, 190 bp for P. minor, 513 bp for P. porosus, and 353 bp for T. obtusus). In silico analysis with 10 other trichodorid species and experimental analysis using samples with these four species, 20 other plant-parasitic and three non-plant-parasitic nematodes demonstrated high specificity with the primers designed. The multiplex PCR amplified desirable fragments using a set of artificially mixed templates containing one, two, three, or four targeted species. The reliability of multiplex PCR results was demonstrated by using nematode populations isolated from infested fields from diverse geographic regions in eight states. The multiplex PCR-based tool developed in this study for the first time provides a simple, rapid, and cost-friendly assay for accurate diagnosis of the four major trichodorid nematodes in the United States.

scholarly journals A Multiplex PCR Assay to Detect and Differentiate Select Agent Strains of Ralstonia solanacearum

Plant Disease ◽  
2015 ◽  
Vol 99 (3) ◽  
pp. 333-341 ◽  
Author(s):  
Michael J. Stulberg ◽  
Jonathan Shao ◽  
Qi Huang
Keyword(s):  
United States ◽  
Multiplex Pcr ◽  
Ralstonia Solanacearum ◽  
Species Complex ◽  
Bacterial Species ◽  
The United States ◽  
Pcr Assay ◽  
Plant Dna ◽  
Multiplex Pcr Assay ◽  
Verification Methods

Ralstonia solanacearum race 3 biovar 2 strains are considered select agents by the U.S. government because they are not endemic to the United States and have the potential to cause brown rot in our potato production fields. Simple and accurate methods are needed for quick identification prior to more discriminating but time-consuming verification methods. We developed a multiplex PCR assay that identifies R. solanacearum species complex strains, signals whether the strain detected is a select agent, and controls for false negatives associated with PCR inhibition or unsuccessful DNA extractions in one reaction. We identified unique sequences of non-phage-related DNA for the R. solanacearum species complex strains, and for select agent strains, using in silico genome subtraction. We also designed and included an internal plant DNA control assay. Our multiplex PCR assay correctly identified 90 R. solanacearum species complex strains and 34 select agent strains, while not recognizing five out-group bacterial species. Additionally, the multiplex PCR assay facilitated the detection of plant DNA and R. solanacearum from infected tomato, potato, geranium, and tobacco plants. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States.

A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites

2018 ◽  
Vol 118 (1) ◽  
pp. 191-201 ◽  
Author(s):  
Arif Ciloglu ◽  
Vincenzo A. Ellis ◽  
Rasa Bernotienė ◽  
Gediminas Valkiūnas ◽  
Staffan Bensch
Keyword(s):  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Pcr Assay ◽  
Haemosporidian Parasites ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
One Step

scholarly journals First Report of the Spiral Nematode Scutellonema brachyurum on Lilyturf in the United States

Plant Disease ◽  
2011 ◽  
Vol 95 (1) ◽  
pp. 74-74 ◽  
Author(s):  
P. Agudelo ◽  
D. Harshman
Keyword(s):  
United States ◽  
Population Density ◽  
The United States ◽  
Parasitic Nematodes ◽  
Initial Population ◽  
Herbaceous Plant ◽  
Plant Parasitic Nematodes ◽  
Essential Information ◽  
First Report ◽  
Plant Parasitic

Lilyturf (Liriope muscari (Decne.) L.H. Bailey), an herbaceous plant, is commonly used in landscaping including borders (along sidewalks, driveways, and trees) and mass plantings as groundcover in the southeastern United States. In December of 2009, a soil sample was submitted to our lab for diagnosis of plant-parasitic nematodes from an area planted with lilyturf located on the Clemson University main campus. A high population density (1,220 individuals/100 cm3 of soil) of spiral nematodes (Scutellonema brachyurum (Steiner, 1938) Andrássy, 1958) was found by routine extraction by sugar centrifugal flotation (3). Other plant-parasitic nematodes, mainly ring nematodes (10 individuals/100 cm3) and stubby root nematodes (10 individuals/100 cm3), were present. To verify if high numbers of spiral nematodes were consistently associated with lilyturf, 20 additional soil and root samples were collected from different places on the campus. In all cases, S. brachyurum was found in densities ranging from 680 to 1,600 individuals/100 cm3 of soil (average of 1,210 individuals/100 cm3). The species was identified by morphological characters of females, including well developed stylet (26 to 30 μm long), no spermatheca, no sperm in uterus, tail broadly rounded with 8 to 12 annules between anus and tail, and scutella at anus level. As is commonly the case for this species, no males were found in any of the samples collected. Examination of the roots revealed numerous, small, reddish brown, necrotic lesions, apparently caused by the feeding and penetration of S. brachyurum. Host plant suitability and pathogenicity of the nematode were tested in the greenhouse. Ten nematode-free lilyturf plants grown individually in 15-cm-diameter plastic pots with pasteurized soil were inoculated with 1,000 spiral nematodes each. Ten uninoculated plants were kept under identical conditions as controls. Three months after inoculation, soil population densities were measured and reproduction factors were calculated to be between 2.8 and 5.4 (final population density divided by initial population density) for the 10 plants. Characteristic lesions previously described were observed in the roots of all inoculated plants, along with slight chlorosis of foliage. These symptoms were not observed on control plants. Spiral nematodes may attack the roots and stolons of lilyturf as ectoparasites or they may enter them and feed in the cortex as endoparasites. Although root lesions were common on affected plants, root injury in general was not severe and generalized root decay was not observed on either the collected plants or those from the greenhouse study. Reports on the pathogenicity of S. brachyurum are variable. Moderate damage was recorded on amaryllis and other ornamentals (4), while measurable damage was observed on tobacco (2), with approximately 100 individuals/100 cm3 of soil, and severe damage on Aloe vera ((L.) Burm. f.), with approximately 500 individuals/100 cm3 (1). To our knowledge, this is the first report of S. brachyurum causing visible symptoms on lilyturf. As the interstate and international movement of perennial plants continues to grow, awareness of the host status of potentially harmful nematodes becomes essential information. References: (1) R. P. Esser et al. Nematropica 16:65, 1986. (2) T. W. Graham. Phytopathology (Abstr.) 45:347, 1955. (3) W. R. Jenkins. Plant Dis. Rep. 48:692, 1964. (4) L. Nong and G. F. Weber. (Abstr.) Phytopathology 54:902, 1964.

scholarly journals Detection of clinically important non tuberculous mycobacteria (NTM) from pulmonary samples through one-step multiplex PCR assay

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Kamal Singh ◽  
Richa Kumari ◽  
Rajneesh Tripathi ◽  
Smita Gupta ◽  
Shampa Anupurba
Keyword(s):  
Multiplex Pcr ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
One Step

Development and validation of a new one step Multiplex-PCR assay for the detection of ten Lactobacillus species

Anaerobe ◽  
2019 ◽  
Vol 59 ◽  
pp. 192-200
Author(s):  
Carlos Gaspar ◽  
Rita Palmeira-de-Oliveira ◽  
José Martinez-de-Oliveira ◽  
José das Neves ◽  
Paula G. Pestana ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Lactobacillus Species ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
One Step ◽  
Development And Validation

scholarly journals Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

2017 ◽  
Vol 55 (9) ◽  
pp. 2736-2751 ◽  
Author(s):  
Hansong Chae ◽  
Seung Jung Han ◽  
Su-Young Kim ◽  
Chang-Seok Ki ◽  
Hee Jae Huh ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Beijing Genotype ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Reliable Technique ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
One Step ◽  
Reference Strains

ABSTRACTThe prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between theMycobacterium tuberculosiscomplex (MTBC) and NTM usingrv0577or RD750, (ii) differentiateM. tuberculosis(M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespreadM. tuberculosisBeijing genotype by targetingmtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium,M. intracellulare,M. abscessus,M. massiliense, andM. kansasii) by targeting IS1311, DT1,mass_3210, andmkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targetedMycobacteriumspecies. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103and 104CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinicalM. tuberculosisand NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC,M. tuberculosis,M. tuberculosisBeijing genotype, and major NTM species.

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