Development and application of a real-time TaqMan® qPCR assay for detection and quantification of ‘Candidatus Mycoplasma haemolamae’ in South American camelids

The U.S. Pacific Northwest states of Oregon and Washington are major producers of cool-season grass seed. Ergot, caused by fungi in the Claviceps purpurea sensu lato group, is an important seed replacement disease of grass worldwide. Microscopic methods that are currently used to quantify airborne Claviceps ascospores captured by spore traps are not currently rapid enough to allow for detecting and reporting of spore numbers in a timely manner, hindering growers from using this information to help manage ergot. We developed a SYBR Green real-time quantitative polymerase chain reaction (qPCR)-based assay for fast and efficient detection and quantification of C. purpurea sensu lato ascospores from Hirst-type spore traps. Species-specificity of the qPCR assay was confirmed against 41 C. purpurea sensu lato isolates collected from six hosts and six other Claviceps spp. Significant relationships were observed between cycle threshold (Ct) values and standard curves of serial dilutions of DNA ranging from 1 pg to 10 ng (R2 = –0.99; P = 0.0002) and DNA extracted from a conidial suspension representing 8 to 80,000 conidia (R2 = –0.99; P = 0.0004). Ct values from qPCR were significantly correlated with results from microscopic examination of spore trap samples from the field (r = –0.68; P < 0.0001) and the procedure was able to detect a single ascospore from spore trap tape samples. The qPCR procedure developed in this study provided a means for quantifying airborne Claviceps ascospores that was highly specific and useful over a wide range of spore densities, and could be performed in a matter of hours instead of days. The qPCR assay developed in this study could be part of an integrated pest management approach to help grass seed growers make risk-based fungicide application decisions for ergot management in grass grown for seed.

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Validation and Application of a Real-Time PCR Assay Based on the CRISPR Array for Serotype-Specific Detection and Quantification of Enterohemorrhagic Escherichia coli O157:H7 in Cattle Feces†

Journal of Food Protection ◽

10.4315/0362-028x.jfp-18-049 ◽

2018 ◽

Vol 81 (7) ◽

pp. 1157-1164 ◽

Cited By ~ 1

Author(s):

LANCE W. NOLL ◽

RACHEL CHALL ◽

PRAGATHI B. SHRIDHAR ◽

XUMING LIU ◽

JIANFA BAI ◽

...

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Shiga Toxin ◽

Culture Method ◽

Qpcr Assay ◽

Escherichia Coli O157 ◽

E Coli ◽

Cattle Feces ◽

Crispr Array ◽

Detection And Quantification

ABSTRACT Several real-time quantitative PCR (qPCR) assays have been developed for detection and quantification of Escherichia coli O157:H7 in complex matrices by targeting genes for serogroup-specific O-antigen (rfbEO157), H7 antigen, and one or more major virulence factors (Shiga toxin and intimin). A major limitation of such assays is that coamplification of H7 and virulence genes in a sample does not signal association of those genes with the O157 serogroup. Clusters of regularly interspaced short palindromic repeats (CRISPR) polymorphisms are highly correlated with certain enterohemorrhagic E. coli (EHEC) serotypes, including O157:H7, and the presence of genes for Shiga toxin (stx1 and stx2) and intimin (eae). Our objectives were to develop and validate a qPCR assay targeting the CRISPR array for the detection and quantification of EHEC O157:H7 in cattle feces and to evaluate the applicability of the assay for detection of and comparison with a four-plex qPCR assay targeting rfbEO157, stx1, stx2, and eae genes and a culture method. Detection limits of the CRISPRO157:H7 qPCR assay for cattle feces spiked with pure cultures were 2.1 × 103 and 2.3 × 100 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n = 576) was compared among the CRISPRO157:H7 qPCR assay, culture method, and four-plex qPCR assay. The CRISPRO157:H7 qPCR detected 42.2% of the samples (243 of 576 samples) as positive for E. coli O157:H7, compared with 30.4% (175 samples) by the culture method. Nearly all samples (97.2%; 560 samples) were positive for rfbEO157 by the four-plex PCR, but 21.8% (122 of 560 samples) were negative for the stx and/or eae genes, making it unlikely that EHEC O157:H7 was present in these samples. Cohen's kappa statistic indicated a fair and poor agreement beyond that due to chance between the CRISPR assay and the culture method and four-plex assay, respectively. This novel qPCR assay can detect the EHEC O157:H7 serotype in cattle feces by targeting CRISPR polymorphisms.

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Developing a real-time PCR assay for direct detection and quantification of Pratylenchus scribneri in field soil

Nematology ◽

10.1163/15685411-00003336 ◽

2020 ◽

Vol 22 (7) ◽

pp. 733-744

Author(s):

Deepika Arora ◽

Guiping Yan ◽

Richard Baidoo

Keyword(s):

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Qpcr Assay ◽

Parasitic Nematodes ◽

Field Soil ◽

Accurate Identification ◽

Soil Dna ◽

Field Soils ◽

Pratylenchus Scribneri ◽

Detection And Quantification

Summary The endomigratory root-lesion nematode, Pratylenchus scribneri, is one of the major plant-parasitic nematodes infecting potato. Accurate identification and quantification of this nematode are essential to develop management strategies but microscopic observations are particularly challenging and time consuming. In this study, a SYBR Green I-based real-time quantitative polymerase chain reaction (qPCR) assay was developed to detect and quantify P. scribneri from field soil DNA extracts. A primer set was designed from the internal transcribed spacer (ITS) region of the P. scribneri rDNA gene. Primer specificity to the target nematode was evaluated by both in silico analysis and qPCR and no detection or non-specific amplification was observed for other non-target nematode species/communities tested in this study. Standard curves were generated using DNA extracts from autoclaved soil infested with varying nematode numbers for calibration. The curves were supported by a high correlation between the P. scribneri numbers artificially added to soil or estimated from naturally infested field soils by traditional methods, and the numbers quantified using the qPCR assay. The assay was able to detect 1 out of 128 (0.0078) equivalents of the DNA of a single nematode in 0.5 g of soil. The qPCR assay developed in this study provides a specific and sensitive detection and quantification of P. scribneri from field soils and a rapid alternative to time-consuming traditional nematode identification and enumeration.

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Detection and Quantification of Rhizoctonia solani AG-1 IA, the Rice Sheath Blight Pathogen, in Rice Using Real-Time PCR

Plant Disease ◽

10.1094/pdis-91-12-1663 ◽

2007 ◽

Vol 91 (12) ◽

pp. 1663-1668 ◽

Cited By ~ 27

Author(s):

Ronald J. Sayler ◽

Yinong Yang

Keyword(s):

Rhizoctonia Solani ◽

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Sheath Blight ◽

Qpcr Assay ◽

Rice Sheath Blight ◽

Internal Transcribed Spacer Region ◽

Causal Organism ◽

Rice Disease ◽

Detection And Quantification

Rhizoctonia solani Kühn is the causal organism of sheath blight, a major rice disease worldwide that severely impairs yield and quality. It is difficult to identify the pathogen in the early phase of the infection and to accurately quantify the fungal development based on visual inspection. Therefore, a rapid and reliable method is advantageous for the detection and quantification of the pathogen causing this important rice disease. In this study, a real-time, quantitative polymerase chain reaction (QPCR) assay was developed to detect and quantify R. solani AG-1 IA DNA from infected rice plants. A specific primer pair was designed based on the internal transcribed spacer region of the fungal ribosomal DNA. The specific detection of R. solani DNA was successful with quantities as low as 1 pg. The QPCR assay could be used for detecting the rice sheath blight pathogen, quantifying fungal aggressiveness, and evaluating the resistance level of rice cultivars.

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Detection and Quantification of Pythium tracheiphilum in Soil by Multiplex Real-Time qPCR

Plant Disease ◽

10.1094/pdis-03-18-0419-re ◽

2019 ◽

Vol 103 (3) ◽

pp. 475-483 ◽

Cited By ~ 6

Author(s):

Hervé Van der Heyden ◽

Thérèse Wallon ◽

C. André Lévesque ◽

Odile Carisse

Keyword(s):

Real Time ◽

Internal Control ◽

Disease Incidence ◽

Quantitative Polymerase Chain Reaction ◽

Pcr Primers ◽

Qpcr Assay ◽

Inoculum Concentration ◽

Soil P ◽

Dry Soil ◽

Detection And Quantification

In Canada, head lettuce (Lactuca sativa capitata) is extensively produced in the muck soils of southwestern Québec. However, yields are increasingly affected by various soilborne pathogens, including Pythium spp., which cause wilt and damping off. In a survey conducted in Québec muck soils in 2010 and 2011, Pythium tracheiphilum Matta was identified as the predominant Pythium sp. in the root of head lettuce showing Pythium stunt symptoms. Therefore, to improve risk assessment and help further understanding of disease epidemiology, a specific and sensitive real-time quantitative polymerase chain reaction (qPCR) assay based on TaqMan-minor groove binder (MGB) technology was developed for P. tracheiphilum. The PCR primers along with a TaqMan-MGB probe were designed from the ribosomal internal transcribed spacer 2 region. A 100-bp product was amplified by PCR from all P. tracheiphilum isolates tested while no PCR product was obtained from 38 other Pythium spp. or from a selection of additional lettuce pathogens tested. In addition to P. tracheiphilum, the assay was multiplexed with an internal control allowing for the individual validation of each PCR. In artificially infested soils, the sensitivity of the qPCR assay was established as 10 oospores/g of dry soil. P. tracheiphilum was not detected in soils in which lettuce has never been grown; however, inoculum ranged from 0 to more than 200,000 oospores/g of dry soil in commercial lettuce fields. Also, disease incidence was positively correlated with inoculum concentration (r = 0.764). The results suggest that inoculum concentration should be considered when making Pythium stunt management decisions. The developed qPCR assay will facilitate reliable detection and quantification of P. tracheiphilum from field soil.

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Detection and Quantification of Phialophora gregata in Soybean and Soil Samples with a Quantitative, Real-Time PCR Assay

Plant Disease ◽

10.1094/pdis-91-6-0736 ◽

2007 ◽

Vol 91 (6) ◽

pp. 736-742 ◽

Cited By ~ 29

Author(s):

D. K. Malvick ◽

A. E. Impullitti

Keyword(s):

Real Time ◽

Single Step ◽

Soil Samples ◽

Qpcr Assay ◽

Taqman Probe ◽

Brown Stem Rot ◽

Phialophora Gregata ◽

Target Dna ◽

Infested Field ◽

Detection And Quantification

Brown stem rot of soybean, caused by the soilborne fungus Phialophora gregata, is a common and widespread disease of soybean (Glycine max) in the midwestern United States. This pathogen is challenging to study due to a long latent period and slow growth. A TaqMan probe-based quantitative, real-time polymerase chain reaction (qPCR) assay was developed for sensitive and specific detection and quantification of genotypes A and B of P. gregata in plant and soil samples. It is sensitive with detection limits of 50 fg of pure genomic DNA, 100 copies of the target DNA sequence, and approximately 400 conidia. The qPCR assay is approximately 1,000 times more sensitive in detecting DNA and conidia of P. gregata, and is more rapid and less sensitive to PCR inhibitors from soybean stems than a standard PCR (sPCR) assay. Using this single-step qPCR assay, low levels of infection were detected in soybean stems at least 1 to 2 weeks prior to symptom development and before P. gregata was detected with sPCR. This assay also was used to detect the pathogen in field-grown plants and in naturally infested field soils. This new qPCR assay is a powerful tool for rapid, specific, and sensitive detection, diagnosis, and quantification of P. gregata in plants and soil, and for advancing studies of the ecology of P. gregata and its interactions with host plants.

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Septicemia and meningoencephalitis caused by Listeria monocytogenes in two neonatal llamas

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638717719479 ◽

2017 ◽

Vol 29 (5) ◽

pp. 700-703 ◽

Cited By ~ 2

Author(s):

Ian K. Hawkins ◽

Marcia Ilha ◽

Eman Anis ◽

Rebecca P. Wilkes

Keyword(s):

Listeria Monocytogenes ◽

Real Time ◽

Real Time Pcr ◽

Pure Culture ◽

Clinical Signs ◽

South American ◽

Tissue Samples ◽

South American Camelids ◽

Autopsy Findings ◽

The Brain

Listeriosis is a disease of humans and domestic mammals (mainly ruminants) with variable manifestations, primarily encephalitis, septicemia, and abortion. Although Listeria monocytogenes readily causes illness in ruminants, the prevalence among domestic South American camelids (llamas and alpacas) is low and has not been documented in their wild counterparts, the vicuna and guanaco. We describe herein the clinical signs, autopsy findings, and histopathology of septicemia and suppurative meningoencephalitis caused by L. monocytogenes in 2 neonatal llamas ( Llama glama) from the same herd. L. monocytogenes was isolated in pure culture and identified by real-time PCR on fresh and paraffin-embedded tissue samples of the brain from both crias. This presentation of septicemic listeriosis with meningoencephalitis in 2 animals from the same group is unusual, especially among llamas.

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Validation of a novel multiplex real-time PCR assay for Trypanosoma cruzi detection and quantification in açai pulp

PLoS ONE ◽

10.1371/journal.pone.0246435 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0246435

Author(s):

Paula Finamore-Araujo ◽

Amanda Faier-Pereira ◽

Carlos Ramon do Nascimento Brito ◽

Eldrinei Gomes Peres ◽

Klenicy Kazumy de Lima Yamaguchi ◽

...

Keyword(s):

Quality Control ◽

Real Time ◽

Dna Extraction ◽

Chagas Disease ◽

Previous History ◽

Global Market ◽

Qpcr Assay ◽

Public Health Perspective ◽

Acai Berry ◽

Detection And Quantification

In Brazil, orally acquired T. cruzi infection has become the most relevant transmission mechanisms from public health perspective. Around 70% of new Chagas disease cases have been associated with consumption of contaminated food or beverages. Açai (Euterpe oleracea and Euterpe precatoria) is currently one of the most commercialized Amazonian fruits in the Brazilian and international markets. Therefore, it has become important to incorporate in the production process some procedures to measure out effective hygiene and product quality control required by global market. Molecular methods have been developed for rapid detection and quantification of T. cruzi DNA in several biological samples, including food matrices, for epidemiological investigation of Chagas disease and food quality control. However, a high-performance molecular methodology since DNA extraction until detection and quantification of T. cruzi DNA in açai berry pulp is still needed. Herein, a simple DNA extraction methodology was standardized from the supernatant of açai berry pulp stabilized in a 6M Guanidine-HCl/0.2M EDTA buffer. In addition, a multiplex real time qPCR assay, targeting T. cruzi DNA and an Exogenous Internal Positive Control was developed and validated, using reference from all T. cruzi DTUs and commercial samples of açai pulp, from an endemic municipality with previous history of oral Chagas disease outbreak. Thus, a high-sensitivity qPCR assay, that could detect up to 0.01 parasite equivalents/mL in açai, was reached. As of the 45 commercial samples analyzed, 9 (20%) were positive for T. cruzi. This high-sensitive, fast, and easy-to-use molecular assay is compatible with most of the laboratories involved in the investigations of oral Chagas disease outbreaks, representing an important tool to the epidemiology, control, and surveillance of Chagas disease.

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Llama and Alpaca Management in Germany—Results of an Online Survey among Owners on Farm Structure, Health Problems and Self-Reflection

Animals ◽

10.3390/ani11010102 ◽

2021 ◽

Vol 11 (1) ◽

pp. 102

Author(s):

Saskia Neubert ◽

Alexandra von Altrock ◽

Michael Wendt ◽

Matthias Gerhard Wagener

Keyword(s):

Online Survey ◽

Animal Husbandry ◽

Health Problems ◽

South American ◽

Wool Production ◽

Self Reflection ◽

South American Camelids ◽

Feeding Management ◽

Management Measures ◽

On Farm

An online survey of llama and alpaca owners was used to collect data on the population, husbandry, feeding, management measures and health problems. A total of 255 questionnaires were evaluated. In total, 55.1% of the owners had started keeping South American camelids within the last six years. The majority managed small farms with fewer than 15 animals (66.4% of 250 farms). More than half of the participants stated that they kept the camelids as hobby animals (64.3%), although they used them for wool production (55.7%) and/or for breeding (51.8%). Vaccination and deworming were carried out on more than 80% of the farms. The most common diseases occurring on the farms were endo- and ectoparasites. A total of 55.3% of the owners estimated their own knowledge of South American camelids as good, 14.5% as very good. In contrast, more than half of the owners agreed little or not at all with the statement that veterinarians generally have sufficient knowledge about South American camelids. Further research is needed to include veterinarians’ perspectives and thus optimise animal husbandry.

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