scholarly journals Development and comparative validation of genomic-driven PCR-based assays to detect Xanthomonas citri pv. citri in citrus plants

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Isabelle Robène ◽  
Véronique Maillot-Lebon ◽  
Aude Chabirand ◽  
Aurélie Moreau ◽  
Nathalie Becker ◽  
...  
Keyword(s):  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Positive Impact ◽  
Transmembrane Protein ◽  
Comparative Genomic ◽  
Analytical Sensitivity ◽  
Bacterial Strains ◽  
Xanthomonas Citri ◽  
Wide Range

Abstract Background Asiatic Citrus Canker, caused by Xanthomonas citri pv. citri, severely impacts citrus production worldwide and hampers international trade. Considerable regulatory procedures have been implemented to prevent the introduction and establishment of X. citri pv. citri into areas where it is not present. The effectiveness of this surveillance largely relies on the availability of specific and sensitive detection protocols. Although several PCR- or real-time PCR-based methods are available, most of them showed analytical specificity issues. Therefore, we developed new conventional and real-time quantitative PCR assays, which target a region identified by comparative genomic analyses, and compared them to existing protocols. Results Our assays target the X. citri pv. citri XAC1051 gene that encodes for a putative transmembrane protein. The real-time PCR assay includes an internal plant control (5.8S rDNA) for validating the assay in the absence of target amplification. A receiver-operating characteristic approach was used in order to determine a reliable cycle cut-off for providing accurate qualitative results. Repeatability, reproducibility and transferability between real-time devices were demonstrated for this duplex qPCR assay (XAC1051-2qPCR). When challenged with an extensive collection of target and non-target strains, both assays displayed a high analytical sensitivity and specificity performance: LOD95% = 754 CFU ml− 1 (15 cells per reaction), 100% inclusivity, 97.2% exclusivity for XAC1051-2qPCR; LOD95% = 5234 CFU ml− 1 (105 cells per reaction), 100% exclusivity and inclusivity for the conventional PCR. Both assays can detect the target from naturally infected citrus fruit. Interestingly, XAC1051-2qPCR detected X. citri pv. citri from herbarium citrus samples. The new PCR-based assays displayed enhanced analytical sensitivity and specificity when compared with previously published PCR and real-time qPCR assays. Conclusions We developed new valuable detection assays useful for routine diagnostics and surveillance of X. citri pv. citri in citrus material. Their reliability was evidenced through numerous trials on a wide range of bacterial strains and plant samples. Successful detection of the pathogen was achieved from both artificially and naturally infected plants, as well as from citrus herbarium samples, suggesting that these assays will have positive impact both for future applied and academic research on this bacterium.

scholarly journals Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

2015 ◽  
Vol 53 (12) ◽  
pp. 3935-3937 ◽  
Author(s):  
Daniel Golparian ◽  
Stina Boräng ◽  
Martin Sundqvist ◽  
Magnus Unemo
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Molecular Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Content Type ◽  
Dna Assay ◽  
Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

scholarly journals Development of a loop-mediated isothermal amplification assay for rapid detection of African swine fever

2020 ◽  
Vol 19 (1) ◽  
pp. 87-100
Author(s):  
Arphaphorn Dokphut ◽  
◽  
Prakit Boonpornprasert ◽  
Tapanut Songkasupa ◽  
Supansa Tangdee ◽  
...  
Keyword(s):  
Early Detection ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
African Swine Fever ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Analytical Sensitivity ◽  
Blue Color ◽  
Loop Mediated Isothermal Amplification

Since the first African swine fever (ASF) outbreak was reported in China in 2018, the disease has spread rapidly to several countries in Asia. The early detection of this disease is essential for the ASF control strategy to be effective. Loop-mediated isothermal amplification (LAMP) is a nucleic acid detection assay that is rapid, simple, cost-effective and field-friendly. In this study, we have developed a colorimetric assay of LAMP to detect ASF virus (ASFV). A set of LAMP primers was designed to target the conserved region of the VP72 gene. The conditions of LAMP were optimized. The amplification products were easily detected by the naked eye using hydroxynaphthol blue (HNB). The positive LAMP reaction generated a violet to sky blue color change. The sensitivity and specificity of LAMP assay were demonstrated in comparison with the OIE-recommended real-time PCR. A total of 211 samples including 121 confiscated pork products and 90 spiked clinical specimens were tested. The optimal amplification of ASFV DNA by LAMP was incubation at 60 °C for 90 min. The analytical sensitivity of ASFV LAMP assay was at least 368 plasmid DNA copies/µL without cross-reactivity with other swine pathogens. The diagnostic sensitivity and specificity of LAMP were 88% and 100%, respectively. There was almost perfect agreement between LAMP and real-time PCR assays (Kappa value=0.84). This novel LAMP assay is deemed to be a rapid, simple, sensitive, specific diagnostic tool and suitable for early detection of ASF to minimize the likelihood of ASF spread nationwide.

scholarly journals Validation of a Novel Diagnostic Approach Combining the VersaTREK™ System for Recovery and Real-Time PCR for the Identification of Mycobacterium chimaera in Water Samples

2021 ◽  
Vol 9 (5) ◽  
pp. 1031
Author(s):  
Roberto Zoccola ◽  
Alessia Di Blasio ◽  
Tiziana Bossotto ◽  
Angela Pontei ◽  
Maria Angelillo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Detection System ◽  
Bacterial Load ◽  
Microbial Control ◽  
Hospital Setting ◽  
Sample Volume ◽  
Analytical Sensitivity ◽  
Initial Water

Mycobacterium chimaera is an emerging pathogen associated with endocarditis and vasculitis following cardiac surgery. Although it can take up to 6–8 weeks to culture on selective solid media, culture-based detection remains the gold standard for diagnosis, so more rapid methods are urgently needed. For the present study, we processed environmental M. chimaera infected simulates at volumes defined in international guidelines. Each preparation underwent real-time PCR; inoculates were placed in a VersaTREK™ automated microbial detection system and onto selective Middlebrook 7H11 agar plates. The validation tests showed that real-time PCR detected DNA up to a concentration of 10 ng/µL. A comparison of the isolation tests showed that the PCR method detected DNA in a dilution of ×102 CFU/mL in the bacterial suspensions, whereas the limit of detection in the VersaTREK™ was <10 CFU/mL. Within less than 3 days, the VersaTREK™ detected an initial bacterial load of 100 CFU. The detection limit did not seem to be influenced by NaOH decontamination or the initial water sample volume; analytical sensitivity was 1.5 × 102 CFU/mL; positivity was determined in under 15 days. VersaTREK™ can expedite mycobacterial growth in a culture. When combined with PCR, it can increase the overall recovery of mycobacteria in environmental samples, making it potentially applicable for microbial control in the hospital setting and also in environments with low levels of contamination by viable mycobacteria.

scholarly journals Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents

2021 ◽  
Vol 9 (8) ◽  
pp. 1610
Author(s):  
Christian Klotz ◽  
Elke Radam ◽  
Sebastian Rausch ◽  
Petra Gosten-Heinrich ◽  
Toni Aebischer
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Gastrointestinal Disease ◽  
Single Copy ◽  
Marker Genes ◽  
Small Rodent ◽  
Analytical Sensitivity ◽  
Single Copy Gene ◽  
Wild Rodents ◽  
Copy Gene

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

scholarly journals Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

2005 ◽  
Vol 71 (7) ◽  
pp. 3911-3916 ◽  
Author(s):  
Mark G. Wise ◽  
Gregory R. Siragusa
Keyword(s):  
Gastrointestinal Tract ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Limit Of Detection ◽  
Quantitative Detection ◽  
Necrotic Enteritis ◽  
Analytical Sensitivity ◽  
Quantitative Real Time Pcr ◽  
Pcr Inhibitor

ABSTRACT Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.

scholarly journals Effect of Feeding Cows Genetically Modified Maize on the Bacterial Community in the Bovine Rumen

2007 ◽  
Vol 73 (24) ◽  
pp. 8012-8017 ◽  
Author(s):  
S. Wiedemann ◽  
P. Gürtler ◽  
C. Albrecht
Keyword(s):  
Bacterial Community ◽  
Real Time ◽  
Real Time Pcr ◽  
Genetically Modified ◽  
Genetically Modified Maize ◽  
Bacterial Strains ◽  
Maize Silage ◽  
Bovine Rumen ◽  
Gm Maize ◽  
Effect Of Feeding

ABSTRACT Rumen-cannulated cows (n = 4) were fed successively silage made from either conventional or genetically modified (GM) maize. Results revealed no effects of GM maize on the dynamics of six ruminal bacterial strains (investigated by real-time PCR) compared to the conventional maize silage.

scholarly journals Comparison of the sensitivity and specificity of real-time PCR and in situ hybridization in HPV16 and 18 detection in archival cervical cancer specimens

2012 ◽  
Vol 50 (2) ◽  
pp. 239-247 ◽  
Author(s):  
Beata Biesaga ◽  
Sława Szostek ◽  
Małgorzata Klimek ◽  
Jerzy Jakubowicz ◽  
Joanna Wysocka
Keyword(s):  
Cervical Cancer ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr

scholarly journals Limited-Resource Preparable Chitosan Magnetic Particles for Extracting Amplification-Ready Zeptomole-Range Nucleic Acid from Complex Biofluid

2021 ◽  
Author(s):  
Sayantan Tripathy ◽  
Arunansu Talukdar ◽  
Goutam Pramanik ◽  
P. V. Rajesh ◽  
Souradyuti Ghosh
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Magnetic Particles ◽  
Limited Resource ◽  
Nucleic Acid Amplification ◽  
Acid Extraction ◽  
Analytical Sensitivity ◽  
Nucleic Acid Extraction ◽  
Spin Column

<b>Layman Summary: </b>Nucleic acid extraction is a key prerequisite for any nucleic acid amplification test (NAAT) or isothermal NAAT (iNAAT) based molecular diagnosis assays.<b> </b>Existing methods utilizes spin column system for nucleic acid extraction which are unsuitable for limited resource settings. Our work explores two methods for chitosan coated magnetic particle preparation that can be executed within 6 h from commonly available chemicals with nothing but a magnetic stirrer and water bath and doable by a minimally trained person. We will also investigated the compatibility of the extracted nucleic acid with downstream NAATs such as real time LAMP, colorimetric LAMP, and real time PCR. In the process, we established the analytical sensitivity of the overall method.<div><br><div><b>Characterization methods</b>: SEM, XRD, EDX, FT-IR</div><div><br></div><div><b>Bioanalytical methods:</b> Real time LAMP, Colorimetric LAMP, Real time PCR</div></div>

scholarly journals The Expression of Ptch1 and Ptch2 in the Stenotic Tissue of Congenital Ureteropelvic Junction Obstruction in Children

2020 ◽  
Author(s):  
Yanfang Yang ◽  
wenwen han ◽  
Weiping Zhang ◽  
Ning Sun
Keyword(s):  
Smooth Muscle ◽  
Western Blot ◽  
Smooth Muscle Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Ureteropelvic Junction Obstruction ◽  
Transmembrane Protein ◽  
Muscle Cells ◽  
Ureteropelvic Junction ◽  
Ureteral Polyps

Abstract Background: Ptch1 and Ptch2 are expressed in tubular epithelium and stromal cells adjacent to the UPJ. They mediate inhibition of Smoothened, a transmembrane protein expressed on the cell surface. If the pathway is disturbed, UPJOcan occur. This aim study aimed to determine the expression of Ptch1 (P1) and Ptch2 (P2) in stenotic segments in children with congenital ureteropelvic junction obstruction (UPJO) compared with normal control subjects. Methods: Stenotic segments of ureter tissues were obtained from 20 UPJO patients.UPJO caused by other pathogenies, such as vessel and ureteral polyps, were excluded. The control ureter specimens were obtained from 10 patients with Wilm’s tumor, and the tissues were confirmed histologically to be unaffected. Immunofluorescence, western blot and real-time PCR were used to investigate the expression of P1 and P2. Statistical methods were used to find the differences between the two groupsResults: P1 and P2 were identified in the cytoplasm of smooth muscle in two groups through immunohistochemistry. However, there were no statistical differences between the two groups in P1 and P2 with immunohistochemistry (P=0.31 and P=0.3, respectively). There were also no statistical differences with western blot (P=0.75 and P=0.9, respectively) and real-time PCR (P=0.52 and P=0.45, respectively). However, with the immunofluorescence it was found that red-stained P1 were diffused in the controls group, but were mainly located in the intracellular perinuclear compartment of smooth muscle cells in UPJO. Conclusions: The expression of P1 and P2 between the two groups had no statistical significant. P1 were mainly located in the intracellular perinuclear compartment of smooth muscle cells in UPJO. The P1 pathway might be disturbed by the abnormal distribution rather than the quantity, which might be one probable pathogenesis of UPJO.

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