scholarly journals Establishment of a duplex real-time qPCR method for detection of Salmonella spp. and Serratia fonticola in fishmeal

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jinghua Ruan ◽  
Wujun Wang ◽  
Tiyin Zhang ◽  
Teng Zheng ◽  
Jing Zheng ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
Bacterial Pathogen ◽  
High Specificity ◽  
Amplification Efficiency ◽  
Salmonella Spp ◽  
Specificity And Sensitivity ◽  
Qpcr Method ◽  
Real Time Qpcr

AbstractSalmonella spp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs. Serratia fonticola is the bacterial species most frequently confused with Salmonella spp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method with invA- and gyrB-specific primers and probes corresponding to Salmonella spp. and S. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection for Salmonella spp. and S. fonticola of 197 copies/μL and 145 copies/μL, respectively (correlation coefficient R2 = 0.999 in both cases). The amplification efficiency for Salmonella spp. and S. fonticola was 98.346% and 96.49%, respectively. Detection of fishmeal was consistent with method GB/T 13091-2018, and all seven artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA from Salmonella spp. and S. fonticola within hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.

scholarly journals Establishment of a Duplex Real-time qPCR Method for Detection of Salmonella Spp. And Serratia Fonticola in Fishmealestablishment of a Duplex Real-time qPCR Method for Detection of Salmonella Spp. And Serratia Fonticola in Fishmeal

2020 ◽  
Author(s):  
Jinghua Ruan ◽  
Wujun Wang ◽  
Tiying Zhang ◽  
Teng Zheng ◽  
Jing Zheng ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
High Specificity ◽  
Clinical Samples ◽  
Amplification Efficiency ◽  
Salmonella Spp ◽  
Specificity And Sensitivity ◽  
Qpcr Method ◽  
Real Time Qpcr

Abstract Salmonella spp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs. Serratiafonticola is the bacterial species most frequently confused with Salmonella spp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method with invA- and gyrB-specific primers and probes corresponding to Salmonella spp. and S. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection for Salmonella spp. and S. fonticola of 197 copies/SL and 145 copies/SL, respectively (correlation coefficient R2 = 0.999 in both cases). The amplification efficiency for Salmonella spp. and S. fonticola was 98.346% and 96.49%, respectively. Detection of clinical samples was consistent with method GB/T 13091-2018, and all seven artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA from Salmonella spp. and S. fonticola within hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.

scholarly journals Establishment and Application of a duplex real-time qPCR method for detection ofSalmonellaspp. andSerratia fonticolain imported feedstuffs

2018 ◽  
Author(s):  
Jing hua Ruan ◽  
Wu jun Wang ◽  
Quan yang Bai ◽  
Ti yin Zhang ◽  
Teng zheng ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Bacterial Species ◽  
High Specificity ◽  
Accurate Method ◽  
Clinical Samples ◽  
Amplification Efficiency ◽  
Specificity And Sensitivity ◽  
Qpcr Method ◽  
Real Time Qpcr

AbstractSalmonellaspp. is a high-risk bacterial pathogen that is monitored in imported animal-derived feedstuffs.Serratia fonticolais the bacterial species most frequently confused withSalmonellaspp. in traditional identification methods based on biochemical characteristics, which are time-consuming and labor-intensive, and thus unsuitable for daily inspection and quarantine work. In this study, we established a duplex real-time qPCR method withinvA-andgyrB-specific primers and probes corresponding toSalmonellaspp. andS. fonticola. The method could simultaneously detect both pathogens in imported feedstuffs, with a minimum limit of detection forSalmonellaspp. andS. fonticolaof 197 copies/μL and 145 copies/μL, respectively (correlation coefficient R2= 0.999 in both cases). The amplification efficiency forSalmonellaspp. andS. fonticolawas 98.346% and 96.49%, respectively. Detection of clinical samples was consistent with method GB/T 13091-2002, and all 20 artificially contaminated imported feed samples were positively identified. Thus, the developed duplex real-time qPCR assay displays high specificity and sensitivity, and can be used for the rapid and accurate detection of genomic DNA fromSalmonellaspp. andS. fonticolawithin hours. This represents a significant improvement in the efficiency of detection of both pathogens in imported feedstuffs.ImportanceImported feedstuffs must be tested for pathogenicSalmonellaspecies that represent a biological hazard. Variousnon-Salmonellacolony-forming species belong toEnterobacteriaceae,andSerratia fonticolaforms colonies of similar color and morphology toSalmonellaspp., leading to confusion in daily quarantine tests. Traditional methods based on biochemical and serological characteristics are cumbersome and labor-intensive, and unable to fully support current quarantine testing demands. Thus, there is an urgent need to develop a rapid and accurate method for the effective identification of these pathogens. The duplex real-time qPCR method established herein can rapidly identifySalmonellaspp. andS. fonticola, and has great potential for application to feed safety and prevention of exterior pathogens.

scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

scholarly journals A Highly Sensitive Cell-Based TLR Reporter Platform for the Specific Detection of Bacterial TLR Ligands

2022 ◽  
Vol 12 ◽  
Author(s):  
Katharina Radakovics ◽  
Claire Battin ◽  
Judith Leitner ◽  
Sabine Geiselhart ◽  
Wolfgang Paster ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Ectopic Expression ◽  
High Sensitivity ◽  
High Specificity ◽  
Reporter System ◽  
Specificity And Sensitivity ◽  
Highly Sensitive ◽  
Definition Of ◽  
The Individual ◽  
Allergen Extracts

Toll-like receptors (TLRs) are primary pattern recognition receptors (PRRs), which recognize conserved microbial components. They play important roles in innate immunity but also in the initiation of adaptive immune responses. Impurities containing TLR ligands are a frequent problem in research but also for the production of therapeutics since TLR ligands can exert strong immunomodulatory properties even in minute amounts. Consequently, there is a need for sensitive tools to detect TLR ligands with high sensitivity and specificity. Here we describe the development of a platform based on a highly sensitive NF-κB::eGFP reporter Jurkat JE6-1 T cell line for the detection of TLR ligands. Ectopic expression of TLRs and their coreceptors and CRISPR/Cas9-mediated deletion of endogenously expressed TLRs was deployed to generate reporter cell lines selectively expressing functional human TLR2/1, TLR2/6, TLR4 or TLR5 complexes. Using well-defined agonists for the respective TLR complexes we could demonstrate high specificity and sensitivity of the individual reporter lines. The limit of detection for LPS was below 1 pg/mL and ligands for TLR2/1 (Pam3CSK4), TLR2/6 (Fsl-1) and TLR5 (flagellin) were detected at concentrations as low as 1.0 ng/mL, 0.2 ng/mL and 10 pg/mL, respectively. We showed that the JE6-1 TLR reporter cells have the utility to characterize different commercially available TLR ligands as well as more complex samples like bacterially expressed proteins or allergen extracts. Impurities in preparations of microbial compounds as well as the lack of specificity of detection systems can lead to erroneous results and currently there is no consensus regarding the involvement of TLRs in the recognition of several molecules with proposed immunostimulatory functions. This reporter system represents a highly suitable tool for the definition of structural requirements for agonists of distinct TLR complexes.

scholarly journals Disposable silicon-based all-in-one micro-qPCR for rapid on-site detection of pathogens

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Estefania Nunez-Bajo ◽  
Alexander Silva Pinto Collins ◽  
Michael Kasimatis ◽  
Yasin Cotur ◽  
Tarek Asfour ◽  
...  
Keyword(s):  
Real Time ◽  
Limit Of Detection ◽  
Low Cost ◽  
High Specificity ◽  
Rapid Screening ◽  
Quantitative Detection ◽  
The Novel ◽  
Novel Coronavirus ◽  
Silicon Based ◽  
Specific Sequences

AbstractRapid screening and low-cost diagnosis play a crucial role in choosing the correct course of intervention when dealing with highly infectious pathogens. This is especially important if the disease-causing agent has no effective treatment, such as the novel coronavirus SARS-CoV-2, and shows no or similar symptoms to other common infections. Here, we report a disposable silicon-based integrated Point-of-Need transducer (TriSilix) for real-time quantitative detection of pathogen-specific sequences of nucleic acids. TriSilix can be produced at wafer-scale in a standard laboratory (37 chips of 10 × 10 × 0.65 mm in size can be produced in 7 h, costing ~0.35 USD per device). We are able to quantitatively detect a 563 bp fragment of genomic DNA of Mycobacterium avium subspecies paratuberculosis through real-time PCR with a limit-of-detection of 20 fg, equivalent to a single bacterium, at the 35th cycle. Using TriSilix, we also detect the cDNA from SARS-CoV-2 (1 pg) with high specificity against SARS-CoV (2003).

scholarly journals Detection and identification of Acanthamoeba and other non-viral causes of infectious keratitis in corneal scrapings by real-time PCR and next-generation sequencing-based 16S-18S gene analysis

2020 ◽  
Author(s):  
Dennis Back Holmgaard ◽  
Celine Barnadas ◽  
Seyed Hossein Mirbarati ◽  
Lee O’Brien Andersen ◽  
Henrik Vedel Nielsen ◽  
...  
Keyword(s):  
Next Generation Sequencing ◽  
Real Time ◽  
Real Time Pcr ◽  
Clinical Relevance ◽  
Bacterial Species ◽  
Screening Method ◽  
Infectious Keratitis ◽  
Next Generation ◽  
Specificity And Sensitivity ◽  
Generation Sequencing

Acanthamoeba is a free-living amoeba of extensive genetic diversity. It may cause infectious keratitis (IK), which can also be caused by bacteria, fungi, and viruses. High diagnostic sensitivity is essential to establish an early diagnosis of Acanthamoeba-associated keratitis. Here, we investigated the applicability of next-generation sequencing (NGS)-based ribosomal gene detection and differentiation (16S-18S) compared with specific real-time PCR for detection of Acanthamoeba. Two hundred DNAs extracted from corneal scrapings and screened by Acanthamoeba-specific real-time PCR were analyzed using an in-house 16S-18S NGS assay. Of these, 24 were positive using specific real-time PCR, 21 of which were positive using the NGS assay. Compared with real-time PCR; the specificity and sensitivity of the NGS assay were 100% and 88%, respectively. Genotypes identified by the NGS assay included T4 (n = 19) and T6 (n = 2). Fungal and bacterial species of potential clinical relevance were identified in 31 of the samples negative for Acanthamoeba, exemplified by Pseudomonas aeruginosa (n = 11), Moraxella spp. (n = 6), Staphylococcus aureus (n = 2), Fusarium spp. (n = 4), and Candida albicans (n = 1). Conclusively, the 16S-18S assay was slightly less sensitive than real-time PCR in detecting Acanthamoeba-specific DNA in corneal scrapings. Robust information on genotype was provided by the NGS assay, and other pathogens of potential clinical relevance were identified in 16% of the samples negative for Acanthamoeba. NGS-based detection of ribosomal genes in corneal scrapings could be an efficient screening method for detecting non-viral causes of IK, including Acanthamoeba.

scholarly journals Development of a Real-Time qPCR Method for Detection and Enumeration of Mycobacterium spp. in Surface Water

2010 ◽  
Vol 76 (21) ◽  
pp. 7348-7351 ◽  
Author(s):  
Nicolas Radomski ◽  
Françoise S. Lucas ◽  
Régis Moilleron ◽  
Emmanuelle Cambau ◽  
Sophie Haenn ◽  
...  
Keyword(s):  
Surface Water ◽  
16S Rrna ◽  
Real Time ◽  
Quantitative Pcr ◽  
Environmental Samples ◽  
Real Time Quantitative Pcr ◽  
Qpcr Method ◽  
Pcr Method ◽  
Real Time Qpcr ◽  
Mycobacterium Spp

ABSTRACT A real-time quantitative PCR method was developed for the detection and enumeration of Mycobacterium spp. from environmental samples and was compared to two other methods already described. The results showed that our method, targeting 16S rRNA, was more specific than the two previously published real-time quantitative PCR methods targeting another 16S rRNA locus and the hsp65 gene (100% versus 44% and 91%, respectively).

scholarly journals Validation of a Non-Specific Dye Real-Time PCR Assay for Porcine Adulteration in Meatball Using ND5 Primer

2017 ◽  
Vol 17 (2) ◽  
pp. 167 ◽  
Author(s):  
Tri Joko Raharjo ◽  
Ery Nourika Alfiraza ◽  
Esti Enjelina ◽  
Deni Pranowo
Keyword(s):  
Real Time ◽  
Annealing Temperature ◽  
Limit Of Detection ◽  
Melting Curve ◽  
High Specificity ◽  
Optimization Method ◽  
Amyl Alcohol ◽  
Melting Curve Analysis ◽  
Rt Pcr ◽  
Temperature Optimization

Porcine adulteration in meatball samples were analyzed using real-time polymerase chain reaction (RT-PCR), based on the ND5 primer obtained by previous study. This work consisted of three stages which were annealing temperature optimization, method validation, and application. DNA template was extracted using phenol-CIAA (chloroform-iso amyl alcohol) method. The optimum annealing temperature for ND5 primers (forward primer 5'-CATTCGCCTCACTCACATTAACC-3' and reverse primer 5'-AAGAGAGAGTTCTACGGTCTGTAG-3') was 58.0 °C, obtained after testing annealing at 50.5 to 59.5 °C gradient temperature with 5 °C interval. Melting curve analysis was done at 65.0 to 95.0 °C, with increasing temperature for 0.5 °C per 2 sec. Method was validated for its specificity, precision and limit of detection. RT-PCR method with ND5 primers produced 227 bp DNA fragment with 78.50 °C Tm value. From eight commercial meatball samples, one was detected containing porcine. The methods showed high specificity and precision, with experimentally determined limits for porcine were no less than 1%.

scholarly journals Antibiogram Study of Bacterial Pathogen from Tilapia Fish in Bangladesh

2019 ◽  
Vol 7 (4) ◽  
pp. 658
Author(s):  
Sume Begum ◽  
Md. Salauddin ◽  
Md. Khaled Hossain ◽  
Mst. Deloara Begum
Keyword(s):  
Oreochromis Niloticus ◽  
Bacterial Species ◽  
Bacterial Pathogen ◽  
Penicillin G ◽  
Local Market ◽  
Salmonella Spp ◽  
Fish Products ◽  
Medical Importance ◽  
Staphylococcus Spp ◽  
Isolated Species

Bacterial pathogens are isolated, identified and antibiogram were performed by taking the skin, gills and intestine of twenty randomly selected Tilapia fish (Oreochromis niloticus) that collected from local market of the Dinajpur city, Bangladesh. A serial dilution was prepared with the stated sample and at the amount of 0.1 ml was plated on nutrient agar, differential also specific media respectively. Then gram’s staining, colony morphology, biochemical test and antibiogram performed respectively. The four different isolated species with frequency of occurrence are 31(40.26%) Escherichia coli, 3 (29.87%) Staphylococcus spp., 13 (16.88%) Pseudomonas spp., 10 (12.99%) Salmonella spp. respectively. Some of these pathogens have tendency to transmit to man, who eat fish or deal with fish and fish products. Amoxicillin, Cefixime, Azithromycin, Chloramphenicol, Ciprofloxacin, Penicillin G, Erythromycin, Vancomycin, Gentamicin and Neomycin antibiotics was performed during sensitivity test. Among the total (77) isolated bacteria were sensitive to gentamycin, chloramphenicol, ciprofloxacin and Azithromycin but resistant to Amoxicillin, Penicillin G, Vancomycin and Erythromycin. The study was conducted in term of medical importance. Hence it is considered that a variety of bacterial species can be associated with fresh Tilapia fish related pathogen to humans.

scholarly journals Comparison of real-time PCR protocols in detection and quantification of fruit tree 16SrX group phytoplasmas

Genetika ◽  
2016 ◽  
Vol 48 (2) ◽  
pp. 629-642 ◽  
Author(s):  
Tomas Kiss ◽  
Tomas Necas ◽  
Jana Necasova
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nested Pcr ◽  
Fruit Tree ◽  
Amplification Efficiency ◽  
Specificity And Sensitivity ◽  
Intercalating Dye ◽  
Detection Characteristics ◽  
Detection And Quantification

In this work, two real-time PCR protocols based on intercalating dye and two on hydrolysis probes were tested using field collected fruit tree samples infected by 16SrX group (AP, PD and ESFY) phytoplasmas. Specificity and sensitivity of protocols and amplification efficiency were the main testing parameters. Results of real-time PCR protocols were compared to nested PCR. All real-time PCR protocols confirmed their specificity of detection. All real-time PCR protocols were 10-100 times more sensitive than nested PCR. Afterall real-time PCR protocols based on hydrolysis probes were 10 times more sensitive than protocols based on intercalating dyes. Among protocols based on hydrolysis probes, slightly better detection characteristics were shown by protocol by CHRISTENSEN et al. (2004).

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