scholarly journals Development of PCR Assays for Diagnosis and Detection of the Pathogens Phacidiopycnis washingtonensis and Sphaeropsis pyriputrescens in Apple Fruit

Plant Disease ◽  
2014 ◽  
Vol 98 (2) ◽  
pp. 241-246 ◽  
Author(s):  
P. Sikdar ◽  
P. Okubara ◽  
M. Mazzola ◽  
C. L. Xiao
Keyword(s):  
Early Detection ◽  
Real Time ◽  
Real Time Pcr ◽  
Apple Fruit ◽  
Correct Identification ◽  
Latent Infections ◽  
Postharvest Diseases ◽  
Internal Transcribed Spacer Region ◽  
Pcr Assays ◽  
Phacidiopycnis Washingtonensis

Speck rot caused by Phacidiopycnis washingtonensis and Sphaeropsis rot caused by Sphaeropsis pyriputrescens are two recently reported postharvest diseases of apple. Infection by these two pathogens occurs in the orchard but remains latent before harvest. Symptoms develop after harvest and are similar to those of gray mold caused by Botrytis cinerea. Accurate diagnosis of these diseases is important during the fruit inspection process, particularly in the instance of fruit destined for export. Early near-harvest detection of latent infections in apple fruit is an important step to implement relevant pre- and postharvest measures for disease control. The aim of this study was to develop polymerase chain reaction (PCR) assays for diagnosis and early detection of latent infections of apple fruit by P. washingtonensis and S. pyriputrescens. Species-specific primers based on the ribosomal DNA internal transcribed spacer region were designed for use in PCR assays. Conventional and real-time PCR assays were developed and validated using fruit inoculated with P. washingtonensis, S. pyriputrescens, or B. cinerea and compared with identifications using traditional isolation-based assays. For wound-inoculated fruit, the PCR assays consistently provided the correct identification of the pathogen used as the inoculant in 6 h of processing time, compared with 5 to 6 days using culture-based methods. Real-time PCR assays effectively detected latent infections in symptomless stem and calyx tissues of fruit that were inoculated with the pathogens in the orchard during the growing season. The PCR assays provide a rapid, accurate method for diagnosis and early detection of these diseases.

scholarly journals Detection and Identification of Six Monilinia spp. Causing Brown Rot Using TaqMan Real-Time PCR from Pure Cultures and Infected Apple Fruit

Plant Disease ◽  
2018 ◽  
Vol 102 (8) ◽  
pp. 1527-1533 ◽  
Author(s):  
Jing-ru Wang ◽  
Li-yun Guo ◽  
Chang-lin Xiao ◽  
Xiao-qiong Zhu
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Related Species ◽  
Severe Disease ◽  
Brown Rot ◽  
Apple Fruit ◽  
Monilinia Fructicola ◽  
The Real ◽  
Pcr Assays ◽  
Monilia Polystroma

Brown rot is a severe disease affecting stone and pome fruit. This disease was recently confirmed to be caused by the following six closely related species: Monilinia fructicola, M. laxa, M. fructigena, Monilia polystroma, M. mumecola, and M. yunnanensis. Because of differences in geographic distributions, some of these species are important quarantine pathogens in certain countries. In this study, we developed TaqMan real-time polymerase chain reaction (PCR) assays to detect and identify the six species. Primer pairs and probes were designed for Monilinia fructicola, M. fructigena, M. laxa, and Monilia polystroma based on sequence differences in the laccase-2 genes. Additionally, based on sequence differences in the elongation factor genes, primer pairs and probes were designed for Monilia mumecola and M. yunnanensis. The real-time PCR assays were able to specifically identify the target pathogens, with detection limits of 10 to 100 fg of DNA, which is equivalent to one to seven conidia. The assays were also able to detect the target pathogens in a mixed DNA sample comprising all six Monilinia spp. and related species. The real-time PCR assays accurately detected target fungi from infected apple fruit. Furthermore, the identification results were consistent with those of traditional morphological methods.

Early Detection of Asian Soybean Rust Using PCR

2006 ◽  
Vol 7 (1) ◽  
pp. 20 ◽  
Author(s):  
Kurt H. Lamour ◽  
Ledare Finley ◽  
Karen L. Snover-Clift ◽  
James P. Stack ◽  
Joy Pierzynski ◽  
...  
Keyword(s):  
Early Detection ◽  
Real Time ◽  
Real Time Pcr ◽  
The United States ◽  
Soybean Rust ◽  
Specific Pcr ◽  
Asian Soybean Rust ◽  
Pcr Assays ◽  
Soybean Plants ◽  
The Impact

Early detection of Asian soybean rust (ASR) is essential to help producers minimize the impact of this serious disease. DNA from ASR-infected soybean plants was used to compare conventional and real-time ASR-specific PCR assays at seven laboratories in the United States. Soybean plants were inoculated with four concentrations of rust spores to establish different levels of infection within the plants. Plant tissue was then harvested at 7 time points over the course of 12 days, and DNA was extracted using two commercially available kits. DNA extracted from soybean plants inoculated with different spore concentrations was tested for ASR using conventional or real-time PCR assays. ASR was consistently detected at 6 days following inoculation with both the conventional and real-time PCR assays. This study demonstrates the ability to reliably detect ASR in soybean before the presence of readily visible symptoms. It also demonstrates the reproducibility of the PCR assay across different real-time PCR platforms at multiple laboratories when the assays were performed in multiple diagnostic laboratories. Accepted for publication 5 April 2006. Published 24 May 2006.

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

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

scholarly journals Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 188
Author(s):  
Tanja Hoffmann ◽  
Andreas Hahn ◽  
Jaco J. Verweij ◽  
Gérard Leboulle ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Acid Extraction ◽  
Nucleic Acid Extraction ◽  
Pcr Assay ◽  
Bead Beating ◽  
Stool Samples ◽  
Pcr Assays ◽  
Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

scholarly journals Comparative Assessment of In-House Real-Time PCRs Targeting Enteric Disease-Associated Microsporidia in Human Stool Samples

Pathogens ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 656
Author(s):  
Konstantin Tanida ◽  
Andreas Hahn ◽  
Kirsten Alexandra Eberhardt ◽  
Egbert Tannich ◽  
Olfert Landt ◽  
...  
Keyword(s):  
Real Time ◽  
Indigenous People ◽  
Real Time Pcr ◽  
Latent Class ◽  
Enterocytozoon Bieneusi ◽  
Enteric Disease ◽  
Immunosuppressed Patients ◽  
Study Population ◽  
Stool Samples ◽  
Pcr Assays

Microsporidiosis is an infection predominantly occurring in immunosuppressed patients and infrequently also in travelers. This study was performed to comparatively evaluate the diagnostic accuracy of real-time PCR assays targeting microsporidia with etiological relevance in the stool of human patients in a latent class analysis-based test comparison without a reference standard with perfect accuracy. Thereby, two one-tube real-time PCR assays and two two-tube real-time PCR assays targeting Enterocytozoon bieneusi and Encephalocytozoon spp. were included in the assessment with reference stool material (20), stool samples from Ghanaian HIV-positive patients (903), and from travelers, migrants and Colombian indigenous people (416). Sensitivity of the assays ranged from 60.4% to 97.4% and specificity from 99.1% to 100% with substantial agreement according to Cohen’s kappa of 79.6%. Microsporidia DNA was detected in the reference material and the stool of the HIV patients but not in the stool of the travelers, migrants, and the Colombian indigenous people. Accuracy-adjusted prevalence was 5.8% (n = 78) for the study population as a whole. In conclusion, reliable detection of enteric disease-associated microsporidia in stool samples by real-time PCR could be demonstrated, but sensitivity between the compared microsporidia-specific real-time PCR assays varied.

scholarly journals Discriminating the eight genotypes of the porcine circovirus type 2 with TaqMan-based real-time PCR

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Ellen Kathrin Link ◽  
Matthias Eddicks ◽  
Liangliang Nan ◽  
Mathias Ritzmann ◽  
Gerd Sutter ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Diagnostic Sensitivity ◽  
Locked Nucleic Acid ◽  
Porcine Circovirus ◽  
Amplification Efficiency ◽  
Pcr Assays

Abstract Background The porcine circovirus type 2 (PCV2) is divided into eight genotypes including the previously described genotypes PCV2a to PCV2f and the two new genotypes PCV2g and PCV2h. PCV2 genotyping has become an important task in molecular epidemiology and to advance research on the prophylaxis and pathogenesis of PCV2 associated diseases. Standard genotyping of PCV2 is based on the sequencing of the viral genome or at least of the open reading frame 2. Although, the circovirus genome is small, classical sequencing is time consuming, expensive, less sensitive and less compatible with mass testing compared with modern real-time PCR assays. Here we report about a new PCV2 genotyping method using qPCR. Methods Based on the analysis of several hundred PCV2 full genome sequences, we identified PCV2 genotype specific sequences or single-nucleotide polymorphisms. We designed six TaqMan PCR assays that are specific for single genotypes PCV2a to PCV2f and two qPCRs targeting two genotypes simultaneously (PCV2g/PCV2d and PCV2h/PCV2c). To improve specific binding of oligonucleotide primers and TaqMan probes, we used locked nucleic acid technology. We evaluated amplification efficiency, diagnostic sensitivity and tested assay specificity for the respective genotypes. Results All eight PCV2 genotype specific qPCRs demonstrated appropriate amplification efficiencies between 91 and 97%. Testing samples from an epidemiological field study demonstrated a diagnostic sensitivity of the respective genotype specific qPCR that was comparable to a highly sensitive pan-PCV2 qPCR system. Genotype specificity of most qPCRs was excellent. Limited unspecific signals were obtained when a high viral load of PCV2b was tested with qPCRs targeting PCV2d or PCV2g. The same was true for the PCV2a specific qPCR when high copy numbers of PCV2d were tested. The qPCR targeting PCV2h/PCV2c showed some minor cross-reaction with PCV2d, PCV2f and PCV2g. Conclusion Genotyping of PCV2 is important for routine diagnosis as well as for epidemiological studies. The introduced genotyping qPCR system is ideal for mass testing and should be a valuable complement to PCV2 sequencing, especially in the case of simultaneous infections with multiple PCV2 genotypes, subclinically infected animals or research studies that require large sample numbers.

Taqman Real-Time PCR Assays for Rapid Detection of Avian PathogenicEscherichia coliIsolates

2014 ◽  
Vol 58 (4) ◽  
pp. 628-631 ◽  
Author(s):  
Nilo Ikuta ◽  
Fabiana de Oliveira Solla Sobral ◽  
Fernanda Kieling Moreira Lehmann ◽  
Vinicius Proença da Silveira ◽  
Silvia de Carli ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Pcr Assays
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