Development and evaluation of a real-time PCR seed lot screening method forFusarium circinatum, causal agent of pitch canker disease

Clavibacter michiganensis subsp. michiganensis (Cmm), the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is quite widely distributed in the EPPO（European and Mediterranean Plant Protection Organization）region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the Cmm pathogen from other closely related subspecies of C. michiganensis, especially the non-pathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital polymerase chain reaction (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of Cmm. This new primers/probe set has been evaluated by qPCRthe real time PCR（qPCR） and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all 5 Cmm strains，and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/mL for both pure Cmm cell suspensions and infected tomato seed, which was 100 times-fold more sensitive than that of the real-time PCR (qPCR ) performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect Cmm even with low bacteria load, which could facilitate both Cmm inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.

Download Full-text

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillus flavus and A. parasiticus in Hazelnut

Toxins ◽

10.3390/toxins12120757 ◽

2020 ◽

Vol 12 (12) ◽

pp. 757

Author(s):

Sara Franco Ortega ◽

Ilenia Siciliano ◽

Simona Prencipe ◽

Maria Lodovica Gullino ◽

Davide Spadaro

Keyword(s):

Food Safety ◽

Real Time ◽

Aspergillus Flavus ◽

Real Time Pcr ◽

Limit Of Detection ◽

Screening Method ◽

Lamp Assay ◽

High Sensitivity ◽

Isothermal Amplification

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

Download Full-text

Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis

Journal of Clinical Microbiology ◽

10.1128/jcm.02224-20 ◽

2020 ◽

Author(s):

Dennis Back Holmgaard ◽

Celine Barnadas ◽

Seyed Hossein Mirbarati ◽

Lee O’Brien Andersen ◽

Henrik Vedel Nielsen ◽

...

Keyword(s):

Next Generation Sequencing ◽

Real Time ◽

Real Time Pcr ◽

Clinical Relevance ◽

Bacterial Species ◽

Screening Method ◽

Infectious Keratitis ◽

Next Generation ◽

Specificity And Sensitivity ◽

Generation Sequencing

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

Download Full-text

Rapid Screening Method for Compounds That Affect the Growth and Germination of Candida albicans, Using a Real-Time PCR Thermocycler

Applied and Environmental Microbiology ◽

10.1128/aem.06227-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 8193-8196 ◽

Cited By ~ 6

Author(s):

Lucja M. Jarosz ◽

Bastiaan P. Krom

Keyword(s):

Candida Albicans ◽

Real Time ◽

Real Time Pcr ◽

Fluorescent Protein ◽

Screening Method ◽

Rapid Screening ◽

Content Type ◽

Wide Range ◽

Hyphal Formation ◽

Green Fluorescent

ABSTRACTWe propose a screening method for compounds affecting growth and germination inCandida albicansusing a real-time PCR thermocycler to quantify green fluorescent protein (GFP) fluorescence. Using PACT1-GFPand PHWP1-GFPreporter strains, the effects of a wide range of compounds on growth and hyphal formation were quantitatively assessed within 3 h after inoculation.

Download Full-text

Potential for Outcrossing in an Apparently Asexual Population of Fusarium circinatum, the Causal Agent of Pitch Canker Disease

Mycologia ◽

10.2307/3761476 ◽

2000 ◽

Vol 92 (6) ◽

pp. 1085 ◽

Cited By ~ 15

Author(s):

Karen Wikler ◽

Thomas R. Gordon ◽

Sharon L. Clark ◽

Michael J. Wingfield ◽

Henriette Britz

Keyword(s):

Causal Agent ◽

Pitch Canker ◽

Fusarium Circinatum ◽

Asexual Population ◽

Canker Disease

Download Full-text

Conventional and real-time pcr assays for detection and identification of rhizoctonia solani AG-2-2, the causal agent of root rot of sugar beet

Pesticidi i fitomedicina ◽

10.2298/pif1901019v ◽

2019 ◽

Vol 34 (1) ◽

pp. 19-29

Author(s):

Mira Vojvodic ◽

Dejan Lazic ◽

Petar Mitrovic ◽

Brankica Tanovic ◽

Ivana Vico ◽

...

Keyword(s):

Sugar Beet ◽

Real Time ◽

Real Time Pcr ◽

Root Rot ◽

Direct Detection ◽

Causal Agent ◽

Future Research ◽

Rhizoctonia Root Rot ◽

Detection And Identification ◽

High Level

Soil-borne fungi belonging to the genus Rhizoctonia are considered to be among the most destructive sugar beet pathogens. Although multinucleate R. solani AG-2-2 is frequently detected as the main causal agent of root rot of sugar beet worldwide, several binucleate (AG-A, AG-E and AG-K) and multinucleate Rhizoctonia (R. solani AG-4, AG-5 and AG-8) have also been included in the disease complex. Due to their soil-borne nature and wide host range, the management of Rhizoctonia root rot of sugar beet is highly demanding. Identification of Rhizoctonia AG associated with root rot of sugar beet is the essential first step in determining a successful disease management strategy. In this paper we report a highly specific and sensitive real-time PCR protocol for detection of R. solani AG-2-2 which showed a high level of specificity after testing against 10 different anastomosis groups and subgroups, including AG-2-1 as the most closely related. Moreover, a similar conventional PCR assay showed the same specificity but proved to be at least a 100 times less sensitive. Future research will include further testing and adaptation of this protocol for direct detection and quantification of R. solani AG-2-2 in different substrates, including plant tissue and soil samples.

Download Full-text