scholarly journals Synergetic effect of non-complementary 5’ AT-rich sequences on the development of a multiplex TaqMan real-time PCR for specific and robust detection of Clavibacter michiganensis and C. michiganensis subsp. nebraskensis

2019 ◽  
Author(s):  
Adriana Larrea-Sarmiento ◽  
Anne M. Alvarez ◽  
James P. Stack ◽  
Mohammad Arif
Keyword(s):  
Real Time ◽  
Abc Transporter ◽  
Real Time Pcr ◽  
High Efficiency ◽  
Synergetic Effect ◽  
The United States ◽  
Comparative Genomic ◽  
Probe Design ◽  
Clavibacter Michiganensis ◽  
Reaction Efficiency

AbstractClavibacter is an agriculturally important genus comprising a single species, Clavibacter michiganensis, and multiple subspecies, including, C. michiganensis subsp. nebraskensis which causes Goss’s wilt/blight of corn and accounts for high yield losses - listed among the five most significant diseases of corn in the United States of America. Our research objective was to develop a robust and rapid multiplex TaqMan real-time PCR (qPCR) to detect C. michiganensis in general and C. michiganensis subsp. nebraskensis with enhanced reliability and accuracy by adding non-complementary AT sequences to the 5’ end of the forward and reverse primers. Comparative genomic analyses were performed to identify unique and conserved gene regions for primer and probe design. The unique genomic regions, ABC transporter ATP-binding protein CDS/ABC-transporter permease and MFS transporter were determined for specific detection of C. michiganensis and C. m. subsp. nebraskensis, respectively. The AT-rich sequences at the 5’ position of the primers enhanced the reaction efficiency and sensitivity of rapid qPCR cycling; the reliability, accuracy and high efficiency of the developed assay was confirmed after testing with 59 strains from inclusivity and exclusivity panels – no false positives or false negatives were detected. The assays were also validated through naturally and artificially infected corn plant samples; all samples were detected for C. michiganensis and C. m. subsp. nebraskensis with 100% accuracy. The assay with 5’ AT-rich sequences detected up to 10- and 100-fg of C. michiganensis and C. michiganensis subsp. nebraskensis genome targets, respectively. No adverse effect was observed when sensitivity assays were spiked with host genomic DNA. Addition of 5’ AT rich sequences enhanced the qPCR reaction efficiency from 0.82 (M = -3.83) and 0.91 (M = -3.54) to 1.04 (with optimum slope value; M = -3.23) for both C. michiganensis and C. michiganensis subsp. nebraskensis, respectively; a increase of 10-fold sensitivity was also obtained with C. michiganensis primer set. The methodology proposed here can be used to optimize the reaction efficiency and to harmonize the diagnostic protocols which have prodigious applications in routine diagnostics, biosecurity and microbial forensics.

scholarly journals Genome Analysis and Development of a Multiplex TaqMan Real-Time PCR for Specific Identification and Detection of Clavibacter michiganensis subsp. nebraskensis

2016 ◽  
Vol 106 (12) ◽  
pp. 1473-1485 ◽  
Author(s):  
James T. Tambong ◽  
Renlin Xu ◽  
Fouad Daayf ◽  
Stephan Brière ◽  
Guillaume J. Bilodeau ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Type Strain ◽  
Draft Genome ◽  
Protein Sequences ◽  
The United States ◽  
Infected Leaf ◽  
Clavibacter Michiganensis ◽  
Blind Test ◽  
Clavibacter Michiganensis Subsp

The reemergence of the Goss’s bacterial wilt and blight disease in corn in the United States and Canada has prompted investigative research to better understand the genome organization. In this study, we generated a draft genome sequence of Clavibacter michiganensis subsp. nebraskensis strain DOAB 395 and performed genome and proteome analysis of C. michiganensis subsp. nebraskensis strains isolated in 2014 (DOAB 397 and DOAB 395) compared with the type strain, NCPPB 2581 (isolated over 40 years ago). The proteomes of strains DOAB 395 and DOAB 397 exhibited a 99.2% homology but had 92.1 and 91.8% homology, respectively, with strain NCPPB 2581. The majority (99.9%) of the protein sequences had a 99.6 to 100% homology between C. michiganensis subsp. nebraskensis strains DOAB 395 and DOAB 397, with only four protein sequences (0.1%) exhibiting a similarity <70%. In contrast, 3.0% of the protein sequences of strain DOAB 395 or DOAB 397 showed low homologies (<70%) with the type strain NCPPB 2581. The genome data were exploited for the development of a multiplex TaqMan real-time polymerase chain reaction (PCR) tool for rapid detection of C. michiganensis subsp. nebraskensis. The specificity of the assay was validated using 122 strains of Clavibacter and non-Clavibacter spp. A blind test and naturally infected leaf samples were used to confirm specificity. The sensitivity (0.1 to 1.0 pg) compared favorably with previously reported real-time PCR assays. This tool should fill the current gap for a reliable diagnostic technique.

Identification of L. casei and L. rhamnosus in the dairy products using Real-Time PCR assay

2015 ◽  
Vol 4 (5) ◽  
pp. 222-225
Author(s):  
K. G. Li ◽  
G. P. Pogossian ◽  
A. K. Moldagulova ◽  
E. E. Bekenova ◽  
A. Abdikadirova ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dairy Products ◽  
Production Control ◽  
High Efficiency ◽  
High Specificity ◽  
Industrial Test ◽  
Fermented Dairy Products ◽  
Species Specific ◽  
Fermented Dairy

  Lactobacilli are essential and important biological objects used in food pro-duction and medicine. One of the sufficient problems is fast, reliable and highly specific identification of lactobacilli in the scientific research and cur-rent production control. We represent two species-specific real-time PCR in the present study to discriminate L. rhamnosus and L. casei basing on the unique peptidoglycan-hydrolase genes p40 and p75 respectively. PCR pri-mers and probes were designed to provide high specificity discrimination via high temperature of PCR annealing stage. High efficiency of the reactions is provided by the size of amplified DNA fragments minimization. Reliable re-producibility of the target sequences amplification and fluorescence detec-tion provide a basis for the future creation of industrial test-systems for op-erational control in the production of fermented dairy products.

scholarly journals A Set of Conventional and Multiplex Real-Time PCR Assays for Direct Detection of Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis in Citrus Fruits

Plant Disease ◽  
2019 ◽  
Vol 103 (2) ◽  
pp. 345-356 ◽  
Author(s):  
Yosra Ahmed ◽  
Jacqueline Hubert ◽  
Céline Fourrier-Jeandel ◽  
Megan M. Dewdney ◽  
Jaime Aguayo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Media ◽  
Direct Detection ◽  
Elongation Factor ◽  
Single Copy ◽  
The United States ◽  
Performance Criteria ◽  
In Planta ◽  
Conventional Pcr

Elsinoë fawcettii, E. australis, and Pseudocercospora angolensis are causal agents of citrus scab and spot diseases. The three pathogens are listed as quarantine pests in many countries and are subject to phytosanitary measures to prevent their entry. Diagnosis of these diseases based on visual symptoms is problematic, as they could be confused with other citrus diseases. Isolation of E. fawcettii, E. australis, and P. angolensis from infected tissues is challenging because they grow slowly on culture media. This study developed rapid and specific detection tools for the in planta detection of these pathogens, using either conventional PCR or one-tube multiplex real-time PCR. Primers and hybridization probes were designed to target the single-copy protein-coding gene MS204 for E. fawcettii and E. australis and the translation elongation factor (Tef-1α) gene for P. angolensis. The specificity of the assays was evaluated by testing against DNA extracted from a large number of isolates (102) collected from different citrus-growing areas in the world and from other hosts. The newly described species E. citricola was not included in the specificity test due to its unavailability from the CBS collection. The detection limits of conventional PCR for the three pathogens were 100, 100, and 10 pg μl−1 gDNA per reaction for E. fawcettii, E. australis, and P. angolensis, respectively. The quadruplex qPCR was fully validated assessing the following performance criteria: sensitivity, specificity, repeatability, reproducibility, and robustness. The quadruplex real-time PCR proved to be highly sensitive, detecting as low as 243, 241, and 242 plasmidic copies (pc) μl−1 of E. fawcettii, E. australis, and P. angolensis, respectively. Sensitivity and specificity of this quadruplex assay were further confirmed using 176 naturally infected citrus samples collected from Ethiopia, Cameroon, the United States, and Australia. The quadruplex assay developed in this study is robust, cost-effective, and capable of high-throughput detection of the three targets directly from citrus samples. This new detection tool will substantially reduce the turnaround time for reliable species identification and allow rapid response and appropriate action.

scholarly journals Sequential Quadriplex Real-Time PCR for Identifying 20 Common emm Types of Group A Streptococcus

2020 ◽  
Vol 59 (1) ◽  
pp. e01764-20
Author(s):  
Srinivasan Velusamy ◽  
Katherine Jordak ◽  
Madeline Kupor ◽  
Sopio Chochua ◽  
Lesley McGee ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Group A Streptococcus ◽  
High Specificity ◽  
The United States ◽  
Rapid Identification ◽  
Invasive Group ◽  
Outbreak Investigations ◽  
Group A ◽  
Culture Negative

ABSTRACTWe developed a sequential quadriplex real-time PCR-based method for rapid identification of 20 emm types commonly found in invasive group A Streptococcus (iGAS) strains recovered through the Centers for Disease Control and Prevention’s Active Bacterial Core surveillance. Each emm real-time PCR assay showed high specificity and accurately identified the respective target emm type, including emm subtypes in the United States. Furthermore, this method is useful for rapid typing of GAS isolates and culture-negative specimens during outbreak investigations.

scholarly journals Performance and Verification of a Real-Time PCR Assay Targeting thegyrAGene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae: TABLE 1

2016 ◽  
Vol 54 (3) ◽  
pp. 805-808 ◽  
Author(s):  
P. Hemarajata ◽  
S. Yang ◽  
O. O. Soge ◽  
R. M. Humphries ◽  
J. D. Klausner
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Real Time Pcr ◽  
The United States ◽  
Test Results ◽  
Pcr Assay ◽  
Content Type ◽  
Agar Dilution ◽  
Ciprofloxacin Resistance ◽  
To Receive

In the United States, 19.2% ofNeisseria gonorrhoeaeisolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive forN. gonorrhoeaeby the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

scholarly journals A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells

2019 ◽  
Vol 57 (9) ◽  
Author(s):  
Qian Wang ◽  
Dimitrios P. Kontoyiannis ◽  
Ruoyu Li ◽  
Wei Chen ◽  
Dingfang Bu ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
High Efficiency ◽  
Gene Mutations ◽  
Health Concern ◽  
Content Type ◽  
Clinical Strains ◽  
Allele Specific ◽  
Resistant Strains

ABSTRACT Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus. The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

scholarly journals Thermodynamically modulated partially double-stranded linear DNA probe design for homogeneous real-time PCR

2007 ◽  
Vol 35 (16) ◽  
pp. e101-e101 ◽  
Author(s):  
S. Huang ◽  
J. Salituro ◽  
N. Tang ◽  
K.-C. Luk ◽  
J. Hackett ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Probe ◽  
Probe Design ◽  
Linear Dna

Evaluation of alternative DNA extraction processes and real-time PCR for detecting Cryptosporidium parvum in drinking water

2015 ◽  
Vol 15 (6) ◽  
pp. 1295-1303
Author(s):  
Gina H. Kimble ◽  
Vincent R. Hill ◽  
James E. Amburgey
Keyword(s):  
Drinking Water ◽  
Real Time ◽  
Dna Extraction ◽  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Real Time Pcr ◽  
Extraction Procedure ◽  
The United States ◽  
Detection Sensitivity ◽  
Spin Column

USEPA Method 1623 is the standard method in the United States for the detection of Cryptosporidium in water samples, but quantitative real-time polymerase chain reaction (qPCR) is an alternative technique that has been successfully used to detect Cryptosporidium in aqueous matrices. This study examined various modifications to a commercial nucleic acid extraction procedure in order to enhance PCR detection sensitivity for Cryptosporidium. An alternative DNA extraction buffer allowed for qPCR detection at lower seed levels than a commercial extraction kit buffer. In addition, the use of a second spin column cycle produced significantly better detection (P = 0.031), and the volume of Tris–EDTA buffer significantly affected crossing threshold values (P = 0.001). The improved extraction procedure was evaluated using 10 L of tap water samples processed by ultrafiltration, centrifugation and immunomagnetic separation. Mean recovery for the sample processing method was determined to be 41% using microscopy and 49% by real-time PCR (P = 0.013). The results of this study demonstrate that real-time PCR can be an effective alternative for detecting and quantifying Cryptosporidium parvum in drinking water samples.

scholarly journals Real-time PCR Quantification and Mycotoxin Production of Fusarium graminearum in Wheat Inoculated with Isolates Collected from Potato, Sugar Beet, and Wheat

2007 ◽  
Vol 97 (7) ◽  
pp. 835-841 ◽  
Author(s):  
Rishi R. Burlakoti ◽  
Rolando Estrada ◽  
Viviana V. Rivera ◽  
Anuradha Boddeda ◽  
Gary A. Secor ◽  
...  
Keyword(s):  
Sugar Beet ◽  
Real Time ◽  
Fusarium Graminearum ◽  
Real Time Pcr ◽  
The United States ◽  
Taqman Probe ◽  
Head Blight ◽  
Dry Rot ◽  
Trichodiene Synthase ◽  
Intergenic Sequences

Fusarium graminearum causes Fusarium head blight (FHB) in small grains worldwide. Although primarily a pathogen of cereals, it also can infect noncereal crops such as potato and sugar beet in the United States. We used a real-time polymerase chain reaction (PCR) method based on intergenic sequences specific to the trichodiene synthase gene (Tri5) from F. graminearum. TaqMan probe and primers were designed and used to estimate DNA content of the pathogen (FgDNA) in the susceptible wheat cv. Grandin after inoculation with the 21 isolates of F. graminearum collected from potato, sugar beet, and wheat. The presence of nine mycotoxins was analyzed in the inoculated wheat heads by gas chromatography and mass spectrometry. All isolates contained the Tri5 gene and were virulent to cv. Grandin. Isolates of F. graminearum differed significantly in virulence (expressed as disease severity), FgDNA content, and mycotoxin accumulation. Potato isolates showed greater variability in producing different mycotoxins than sugar beet and wheat isolates. Correlation analysis showed a significant (P < 0.001) positive relationship between FgDNA content and FHB severity or deoxynivalenol (DON) production. Moreover, a significant (P < 0.001) positive correlation between FHB severity and DON content was observed. Our findings revealed that F. graminearum causing potato dry rot and sugar beet decay could be potential sources of inoculum for FHB epidemics in wheat. Real-time PCR assay provides sensitive and accurate quantification of F. graminearum in wheat and can be useful for monitoring the colonization of wheat grains by F. graminearum in controlled environments, and evaluating wheat germplasms for resistance to FHB.

Sign in / Sign up

Export Citation Format

Share Document