scholarly journals Improved Primers for the Specific Detection of Leifsonia xyli subsp. xyli in Sugarcane Using a Conventional PCR Assay

Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3251-3258
Author(s):  
Sheng-Ren Sun ◽  
Jun-Lü Chen ◽  
Yao-Yao Duan ◽  
Na Chu ◽  
Mei-Ting Huang ◽  
...  
Keyword(s):  
Pathogen Detection ◽  
Dna Amplification ◽  
High Yield ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Saccharum Spp ◽  
Sensitivity Tests ◽  
Field Samples ◽  
Pcr Assays ◽  
Higher Sensitivity

Ratoon stunting disease (RSD), one of the most important diseases of sugarcane, is caused by the bacterium Leifsonia xyli subsp. xyli (Lxx). Lxx infects sugarcane worldwide and RSD results in high yield losses and varietal degeneration. It is highly challenging to diagnose RSD based on visual symptomatology because this disease does not exhibit distinct external and internal symptoms. In this study, a novel Lxx-specific primer pair Lxx-F1/Lxx-R1 was designed to detect this pathogen using a conventional PCR assay. These primers were then compared with four published Lxx-specific primers and one universal Leifsonia generic primer pair LayF/LayR. Sugarcane leaf samples were collected from Saccharum spp. hybrids in commercial fields (315 samples) and from germplasm clones of five Saccharum species and Erianthus arundinaceus (216 samples). These samples were used for comparative field diagnosis with six conventional PCR assays. Sensitivity tests suggested that the PCR assay with primers Lxx-F1/Lxx-R1 had the same detection limit (1 pg of Lxx genomic DNA) as the primer pairs Cxx1/Cxx2 and CxxITSf#5/CxxITSr#5 and had 10-fold higher sensitivity than the primer pairs Pat1-F2/Pat1-R2, LayF/LayR, and C2F/C2R. Comparison of PCR assays revealed that natural Lxx-infection incidence (6.1%) in field sample evaluation identified by Lxx-F1/Lxx-R1 primers was higher than incidences (0.7 to 3.0%) determined by other primer pairs. Moreover, no nonspecific DNA amplification occurred within these field samples with Lxx-F1/Lxx-R1 primers, unlike with the primer pairs Cxx1/Cxx2 and LayF/LayR. Diverse Leifsonia strains were identified by PCR detection with LayF/LayR primers in the field samples, whereas whether these Leifsonia strains were pathogenic to sugarcane requires further research. Our investigations revealed that the PCR assay with the newly designed primers Lxx-F1/Lxx-R1 could be widely used for RSD diagnosis and Lxx-pathogen detection with satisfactory sensitivity and specificity.

scholarly journals Development of Conventional and Real-Time PCR Assays for the Rapid Detection of Group B Streptococci

2000 ◽  
Vol 46 (3) ◽  
pp. 324-331 ◽  
Author(s):  
Danbing Ke ◽  
Christian Ménard ◽  
François J Picard ◽  
Maurice Boissinot ◽  
Marc Ouellette ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Culture Method ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Group B Streptococci ◽  
Standard Culture ◽  
Pcr Assays ◽  
Group B

Abstract Background: Group B streptococci (GBS), or Streptococcus agalactiae, are the leading bacterial cause of meningitis and bacterial sepsis in newborns. Currently available rapid methods to detect GBS from clinical specimens are unsuitable for replacement of culture methods, mainly because of their lack of sensitivity. Methods: We have developed a PCR-based assay for the rapid detection of GBS. The cfb gene encoding the Christie-Atkins-Munch-Petersen (CAMP) factor was selected as the genetic target for the assay. The PCR primers were initially tested by a conventional PCR method followed by gel electrophoresis. The assay was then adapted for use with the LightCyclerTM. For this purpose, two fluorogenic adjacent hybridization probes complementary to the GBS-specific amplicon were designed and tested. In addition, a rapid sample-processing protocol was evaluated by colony-forming unit counting and PCR. A total of 15 vaginal samples were tested by both standard culture method and the two PCR assays. Results: The conventional PCR assay was specific because it amplified only GBS DNA among 125 bacterial and fungal species tested, and was able to detect all 162 GBS isolates from various geographical areas. This PCR assay allowed detection of as few as one genome copy of GBS. The real-time PCR assay was comparable to conventional PCR assay in terms of sensitivity and specificity, but it was more rapid, requiring only ∼30 min for amplification and computer-based data analysis. The presence of vaginal specimens had no detrimental effect on the sensitivity of the PCR with the sample preparation protocol used. All four GBS-positive samples identified by the standard culture method were detected by the two PCR assays. Conclusion: These assays provide promising tools for the rapid detection and identification of GBS.

scholarly journals PARASITOLOGICAL AND MOLECULAR DIAGNOSIS IN EXPERIMENTAL Strongyloides venezuelensis INFECTION

2013 ◽  
Vol 55 (2) ◽  
pp. 141-143 ◽  
Author(s):  
Fabiana Martins Paula ◽  
Renata Barnabé Sitta ◽  
Fernanda Mello Malta ◽  
Maiara Gottardi ◽  
Marcelo Andreetta Corral ◽  
...  
Keyword(s):  
Molecular Diagnosis ◽  
Rattus Norvegicus ◽  
Parasitic Nematode ◽  
Pcr Assay ◽  
Fecal Samples ◽  
Strongyloides Venezuelensis ◽  
Pcr Assays ◽  
Higher Sensitivity

Strongyloides venezuelensis is a parasitic nematode of rats which is frequently used as a model to study human and animal strongyloidiasis. The aim of this study was to evaluate the correlation between parasitological and molecular diagnosis in Strongyloides venezuelensis infection. PCR assays were used to detect S. venezuelensis DNA in fecal samples obtained from experimentally infected Rattus norvegicus. The results showed a higher sensitivity of the PCR assay in detecting the infection compared to parasitological methods.

scholarly journals Rapid Screening for SARS-CoV-2 Variants of Concern in Clinical and Environmental Samples Using Nested RT-PCR Assays Targeting Key Mutations of the Spike Protein

2021 ◽  
Author(s):  
G. La Rosa ◽  
P. Mancini ◽  
G. Bonanno Ferraro ◽  
C. Veneri ◽  
M. Iaconelli ◽  
...  
Keyword(s):  
Spike Protein ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Correct Identification ◽  
Potential Contribution ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Field Samples ◽  
Pcr Assays ◽  
Viral Isolates

SUMMARYNew SARS-CoV-2 mutations are constantly emerging, raising concerns of increased transmissibility, virulence or escape from host immune response.We describe a nested RT-PCR assay (∼1500 bps) to detect multiple key spike protein mutations distinctive of the major known circulating SARS-CoV-2 variants, including the three Variants of Concern (VOCs) 20I/501Y.V1 (United Kingdom), 20H/501Y.V2 (South Africa), and 20J/501Y.V3 (Brazil), as well as the 20E.EU1 variant (Spain), the CAL.20C recently identified in California, and the mink-associated variant (GR, lineage B.1.1.298). Prior to application to field samples, the discriminatory potential of this PCR assay was explored using GISAID and Nextclade. To extend variant detection to challenging matrices such as sewage, where the amplification of long fragments is problematic, two short nested RT-PCR assays (∼300 bps) were also designed, targeting portions of the region spanned by the long nested assay.The three newly-designed assays were then tested on field samples, including 7 fully-sequenced viral isolates from swab samples and 34 urban wastewater samples, some of which collected in areas where circulation of VOCs had been reported.The long assay successfully amplified all the previously characterized viral isolates, allowing the correct identification of variants 20I/501Y.V1 and 20E.EU1 present in the panel. The sequences obtained using the short assays were consistent with those obtained with the long assay. Mutations characteristic of VOCs (UK and Brazilian variant) and of other variant (Spanish) were detected in sewage samples. To our knowledge, this is the first evidence of the presence of sequences harboring key mutations of 20I/501Y.V1 and 20J/501Y.V3 in urban wastewaters, highlighting the potential contribution of wastewater surveillance to explore SARS-CoV-2 diversity.The developed nested RT-PCR assays can be used as an initial rapid screening test to select clinical samples containing mutations of interest. This can speed up diagnosis and optimize resources since it allows full genome sequencing to be done only on clinically relevant specimens. The assays can be also employed for a rapid and cost-effective detection of VOCs or other variants in sewage for the purposes of wastewater-based epidemiology. The approach proposed here can be used to better understand SARS-CoV-2 variant diversity, geographic distribution and impact worldwide.

scholarly journals Choice of molecular assay determines ranavirus detection probability and inferences about prevalence and occurrence

2020 ◽  
Vol 141 ◽  
pp. 139-147
Author(s):  
FJ Wynne ◽  
R Puschendorf ◽  
ME Knight ◽  
SJ Price
Keyword(s):  
Population Declines ◽  
Molecular Assay ◽  
Conventional Pcr ◽  
Emerging Pathogens ◽  
Pcr Assay ◽  
Molecular Assays ◽  
Field Samples ◽  
Assay Precision ◽  
Viral Lineage ◽  
Inter Assay Variation

Ranaviruses are emerging pathogens that can cause morbidity, mortality and population declines in ectothermic hosts; however, there is no standardized approach to diagnostics. Here, we compared the inter-assay variation and intra-assay precision among 2 commonly used quantitative PCRs (qPCRs), a conventional and a nested PCR assay (used as a gold standard), using laboratory-propagated ranavirus (FV3 and CMTV) and field-collected samples. A qPCR assay (‘Leung’) detected viral DNA in dilutions 2 orders of magnitude lower than other assays regardless of the viral lineage of the cultured isolate (FV3/CMTV). The second qPCR (‘Brunner’) was slightly more sensitive than the conventional PCR (‘Mao’ assay). For field samples, the Leung qPCR detected all known positives, while the Mao assay PCR only detected 2.5% of the positive samples. Amplicon sequences from the 2 conventional PCRs were shown to be useful for inferring viral lineage. Inaccurate results will bias estimates of the distribution and prevalence of ranaviruses, and together these findings emphasize that molecular assays should be chosen carefully in the context of study aims.

scholarly journals Early Detection of Leifsonia xyli subsp. xyli in Sugarcane Leaves by Real-Time Polymerase Chain Reaction

Plant Disease ◽  
2007 ◽  
Vol 91 (4) ◽  
pp. 430-434 ◽  
Author(s):  
M. P. Grisham ◽  
Y.-B. Pan ◽  
E. P. Richard
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Close Agreement ◽  
Leaf Tissue ◽  
Conventional Pcr ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Polymerase Chain ◽  
Pcr Assays

A real-time, polymerase chain reaction (PCR) assay was developed for detecting Leifsonia xyli subsp. xyli in sugarcane leaf tissue. Real-time PCR assays were conducted on the youngest, fully expanded leaf of three cultivars collected bi-weekly from field nurseries between 11 April and 19 July 2005. L. xyli subsp. xyli infection was detected in leaves collected at all sampling dates, including those from 1-month-old plants on 11 April. Assays conducted on older, more rapidly growing plants (28 July and 21 October 2005) indicated that leaf position affects assay efficiency. Conventional PCR was less efficient than real-time PCR for detecting L. xyli subsp. xyli in leaf tissue. Real-time PCR was used to rank cultivars for susceptibility to L. xyli subsp. xyli infection based on the relative titer of L. xyli subsp. xyli in leaves of inoculated, 3- and 4-month-old greenhouse-grown plants. The ranking of cultivars by real-time PCR was in close agreement with the ranking determined by tissue-blot enzyme immunoassay performed on tissue from 7- to 9-month-old stalks.

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.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

scholarly journals Extreme PCR: Efficient and Specific DNA Amplification in 15–60 Seconds

2015 ◽  
Vol 61 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Jared S Farrar ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
High Resolution Melting ◽  
Dna Amplification ◽  
Temperature Cycling ◽  
High Yield ◽  
Temperature Cycle ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Agarose Gels

Abstract BACKGROUND PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown. METHODS We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4–2.0 s at annealing/extension temperatures of 62 °C–76 °C and denaturation temperatures of 85 °C–92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring. RESULTS Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15–60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%–95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles. CONCLUSIONS The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4–2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

scholarly journals Mitigating Future Respiratory Virus Pandemics: New Threats and Approaches to Consider

Viruses ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 637
Author(s):  
Gregory C. Gray ◽  
Emily R. Robie ◽  
Caleb J. Studstill ◽  
Charles L. Nunn
Keyword(s):  
Pathogen Detection ◽  
Respiratory Virus ◽  
Emerging Infectious Diseases ◽  
Health Resources ◽  
Mitigation Strategies ◽  
High Yield ◽  
Animal Virus ◽  
Virus Surveillance ◽  
Human Animal ◽  
Cost Efficient

Despite many recent efforts to predict and control emerging infectious disease threats to humans, we failed to anticipate the zoonotic viruses which led to pandemics in 2009 and 2020. The morbidity, mortality, and economic costs of these pandemics have been staggering. We desperately need a more targeted, cost-efficient, and sustainable strategy to detect and mitigate future zoonotic respiratory virus threats. Evidence suggests that the transition from an animal virus to a human pathogen is incremental and requires a considerable number of spillover events and considerable time before a pandemic variant emerges. This evolutionary view argues for the refocusing of public health resources on novel respiratory virus surveillance at human–animal interfaces in geographical hotspots for emerging infectious diseases. Where human–animal interface surveillance is not possible, a secondary high-yield, cost-efficient strategy is to conduct novel respiratory virus surveillance among pneumonia patients in these same hotspots. When novel pathogens are discovered, they must be quickly assessed for their human risk and, if indicated, mitigation strategies initiated. In this review, we discuss the most common respiratory virus threats, current efforts at early emerging pathogen detection, and propose and defend new molecular pathogen discovery strategies with the goal of preempting future pandemics.

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