Detection and identification of pathogenic dermatophytes using multiplex real-time PCR assays

Meloidogyne enterolobii is a quarantine root-knot nematode posing a major threat to agricultural production systems worldwide. It attacks many host plants, including important agricultural crops, ornamentals, and trees. M. enterolobii is a highly virulent and pathogenic root-knot nematode species, able to reproduce on plants resistant to other Meloidogyne spp. Significant crop damage has been reported in Asia, South America, Africa, the United States, France, and greenhouses in Switzerland. To identify potential introduction pathways and ensure appropriate phytosanitary measures and management strategies, accurate detection and identification tools are needed. Therefore, two real-time quantitative polymerase chain reaction (PCR) assays based on the second intergenic spacer region of the ribosomal DNA cistron and the cytochrome oxidase c subunit I (COI) gene using locked nucleic acid probes were developed and validated for fast and reliable detection and identification of M. enterolobii. Analytical specificity was confirmed with 16 M. enterolobii populations, 16 populations of eight closely related Meloidogyne spp., and four species from other nematode genera. Optimizing and testing the assays on two real-time PCR platforms revealed an analytical sensitivity of one juvenile in a background of 1,000 nematodes and the intended limit of detection of one juvenile per 100 ml of soil. Both assays performed equally well, with the COI-based assay showing a slightly better performance concerning detection of M. enterolobii target DNA in complex DNA backgrounds.

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Development of Real-Time and Conventional PCR Assays for Identifying Stubby Root Nematode Paratrichodorus allius

Plant Disease ◽

10.1094/pdis-10-16-1431-re ◽

2017 ◽

Vol 101 (6) ◽

pp. 964-972 ◽

Cited By ~ 6

Author(s):

Danqiong Huang ◽

Guiping Yan ◽

Andrea M. Skantar

Keyword(s):

Real Time ◽

Molecular Diagnosis ◽

Real Time Pcr ◽

Tobacco Rattle Virus ◽

Nematode Species ◽

Detection Sensitivity ◽

Conventional Pcr ◽

Nematode Communities ◽

Detection And Identification ◽

Pcr Assays

Paratrichodorus allius is an important pest on many crops, particularly on potato due to its ability to transmit Tobacco rattle virus causing corky ringspot disease on tubers. Detection and identification of P. allius are important for effective disease management. In this study, a rapid and reliable molecular diagnosis of this nematode targeting internal transcribed spacer ribosomal DNA was established. The specificity of the designed primers was evaluated using 29 nematode species and results showed that a single amplicon was produced from DNA of P. allius only. Detection sensitivity analysis indicated that a 9.6 × 10−4 ng of DNA template could be detected by conventional PCR and 1.92 × 10−4 ng of DNA by real-time PCR. The PCR assays amplified DNA of stubby root nematodes isolated from 18 soil samples in North Dakota and Minnesota, which were confirmed as P. allius by sequencing. Both conventional PCR and real-time PCR assays amplified target nematodes from complex nematode communities, supporting the success of this molecular diagnosis of P. allius. This is the first report of P. allius identification using the real-time PCR method and from nematode communities with other nematodes using conventional PCR. The new PCR assays provide rapid species identification and are suitable for use in diagnostic laboratories and detection of field infestations with P. allius.

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Real-Time PCR Detection and Identification of Prohibited Mammalian and Avian Material in Animal Feeds

Journal of Food Protection ◽

10.4315/0362-028x-72.5.1055 ◽

2009 ◽

Vol 72 (5) ◽

pp. 1055-1062 ◽

Cited By ~ 19

Author(s):

SAIRA CAWTHRAW ◽

GINNY C. SAUNDERS ◽

TREVOR C. MARTIN ◽

JASON SAWYER ◽

OTTO WINDL ◽

...

Keyword(s):

16S Rrna ◽

Real Time ◽

Real Time Pcr ◽

Animal Feed ◽

Meat And Bone Meal ◽

Bone Meal ◽

Detection And Identification ◽

Pcr Assays ◽

Species Specific ◽

Feed Samples

A method for the detection and identification of “prohibited” mammalian or avian material in animal feed was developed and assessed through the analysis of DNA. A generic real-time PCR assay was designed to detect the presence of mammalian and avian mitochondrial DNA 16S rRNA genes in animal feed samples. Samples positive with this screening method were further investigated using identification assays to detect the 16S rRNA gene from bovine, ovine, porcine, and avian species and to determine whether the DNA originated from species whose material is prohibited from inclusion in farmed animal feed. An internal positive control was coamplified in the 16S real-time PCR assays to monitor PCR amplification efficiency and avoid potential false-negative results. Using vegetable-based feed standards spiked with meat and bone meal generated with a commercial rendering process, 0.1% meat and bone meal could be detected using the general and species-specific 16S assays. The species-specific assays had 100% specificity for the homologous target species. The 16S real-time PCR assays were evaluated alongside existing tests based on protein evaluation or microscopic examination for a wide range of commercial animal feed samples. In total, 111 (0.76%) of 14,678 samples examined contained prohibited material based on the results from at least one of these tests. However, most positive results did not represent noncompliance because they were associated with samples of pet food, which can legitimately contain material prohibited for use in food for farmed animals. The species-specific 16S assays confirmed the presence of prohibited material in 75% of the 111 samples, whereas the existing protein and microscope tests confirmed the presence of this material in 25 and 54% of the samples, respectively.

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Multiplex real‐time PCR assays for the detection and identification of Heterobasidion species attacking conifers in Europe

Plant Pathology ◽

10.1111/ppa.13071 ◽

2019 ◽

Vol 68 (8) ◽

pp. 1493-1507 ◽

Cited By ~ 2

Author(s):

R. Ioos ◽

P. Chrétien ◽

J. Perrault ◽

C. Jeandel ◽

C. Dutech ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Detection And Identification ◽

Pcr Assays

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DEVELOPMENT OF MULTIPLEX REAL-TIME PCR ASSAY FOR SIX PATHOGENS: RAPID TOOL FOR DIAGNOSIS OF BACTERIAL MENINGITIS

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2019.3.8 ◽

2019 ◽

pp. 60-66

Author(s):

Viet Quynh Tram Ngo ◽

Thi Ti Na Nguyen ◽

Hoang Bach Nguyen ◽

Thi Tuyet Ngoc Tran ◽

Thi Nam Lien Nguyen ◽

...

Keyword(s):

Bacterial Meningitis ◽

Real Time ◽

Real Time Pcr ◽

Diagnostic Methods ◽

Type B ◽

E Coli ◽

Pcr Assays ◽

Set Up ◽

Etiological Agents ◽

Suis Serotype

Introduction: Bacterial meningitis is an acute central nervous infection with high mortality or permanent neurological sequelae if remained undiagnosed. However, traditional diagnostic methods for bacterial meningitis pose challenge in prompt and precise identification of causative agents. Aims: The present study will therefore aim to set up in-house PCR assays for diagnosis of six pathogens causing the disease including H. influenzae type b, S. pneumoniae, N. meningitidis, S. suis serotype 2, E. coli and S. aureus. Methods: inhouse PCR assays for detecting six above-mentioned bacteria were optimized after specific pairs of primers and probes collected from the reliable literature resources and then were performed for cerebrospinal fluid (CSF) samples from patients with suspected meningitis in Hue Hospitals. Results: The set of four PCR assays was developed including a multiplex real-time PCR for S. suis serotype 2, H. influenzae type b and N. meningitides; three monoplex real-time PCRs for E. coli, S. aureus and S. pneumoniae. Application of the in-house PCRs for 116 CSF samples, the results indicated that 48 (39.7%) cases were positive with S. suis serotype 2; one case was positive with H. influenzae type b; 4 cases were positive with E. coli; pneumococcal meningitis were 19 (16.4%) cases, meningitis with S. aureus and N. meningitidis were not observed in any CSF samples in this study. Conclusion: our in-house real-time PCR assays are rapid, sensitive and specific tools for routine diagnosis to detect six mentioned above meningitis etiological agents. Key words: Bacterial meningitis, etiological agents, multiplex real-time PCR

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