scholarly journals Design and validation of oligonucleotide primers suitable for waterborne bacterial pathogen detection via real-time qPCR

2021 ◽  
Author(s):  
Shawn Thomas Clark

Fecal coliforms have been used as indicators to evaluate health risks associated with the microbiological quality of water for many years. Recent studies have challenged their ability to accurately predict bacterial numbers in the natural environment. DNA-based assays are proposed candidates to replace existing methods, but protocols suited for standardized direct-use have not yet been sufficiently developed. The objective of this study was to examine the feasibility of using real-time quantitative PCR (qPCR) to detect contamination from five waterborne bacterial pathogens in surface and treated drinking waters. Robust oligonucleotide primers were assembled to target virulence-associated genes. Primers were found to have high specificity and increased sensitivity for low pathogen loads of 10 cells/mL, as determined experimentally via qPCR. Detection of pathogenic cells directly from an environmental matrix has also been demonstrated using a filtration-extraction procedure. The developed protocols have shown their potential for use in conjunction with traditional indicator techniques.

2021 ◽  
Author(s):  
Shawn Thomas Clark

Fecal coliforms have been used as indicators to evaluate health risks associated with the microbiological quality of water for many years. Recent studies have challenged their ability to accurately predict bacterial numbers in the natural environment. DNA-based assays are proposed candidates to replace existing methods, but protocols suited for standardized direct-use have not yet been sufficiently developed. The objective of this study was to examine the feasibility of using real-time quantitative PCR (qPCR) to detect contamination from five waterborne bacterial pathogens in surface and treated drinking waters. Robust oligonucleotide primers were assembled to target virulence-associated genes. Primers were found to have high specificity and increased sensitivity for low pathogen loads of 10 cells/mL, as determined experimentally via qPCR. Detection of pathogenic cells directly from an environmental matrix has also been demonstrated using a filtration-extraction procedure. The developed protocols have shown their potential for use in conjunction with traditional indicator techniques.


2010 ◽  
Vol 77 (1) ◽  
pp. 89-97 ◽  
Author(s):  
Ana Palacio-Bielsa ◽  
Jaime Cubero ◽  
Miguel A. Cambra ◽  
Raquel Collados ◽  
Isabel M. Berruete ◽  
...  

ABSTRACTXanthomonas arboricolapv. pruni, the causal agent of bacterial spot disease of stone fruit, is considered a quarantine organism by the European Union and the European and Mediterranean Plant Protection Organization (EPPO). The bacterium can undergo an epiphytic phase and/or be latent and can be transmitted by plant material, but currently, only visual inspections are used to certify plants as beingX. arboricolapv. pruni free. A novel and highly sensitive real-time TaqMan PCR detection protocol was designed based on a sequence of a gene for a putative protein related to an ABC transporter ATP-binding system inX. arboricolapv. pruni. Pathogen detection can be completed within a few hours with a sensitivity of 102CFU ml−1, thus surpassing the sensitivity of the existing conventional PCR. Specificity was assessed forX. arboricolapv. pruni strains from different origins as well as for closely relatedXanthomonasspecies, non-Xanthomonasspecies, saprophytic bacteria, and healthyPrunussamples. The efficiency of the developed protocol was evaluated with field samples of 14Prunusspecies and rootstocks. For symptomatic leaf samples, the protocol was very efficient even when washed tissues of the leaves were directly amplified without any previous DNA extraction. For samples of 117 asymptomatic leaves and 285 buds, the protocol was more efficient after a simple DNA extraction, andX. arboricolapv. pruni was detected in 9.4% and 9.1% of the 402 samples analyzed, respectively, demonstrating its frequent epiphytic or endophytic phase. This newly developed real-time PCR protocol can be used as a quantitative assay, offers a reliable and sensitive test forX. arboricolapv. pruni, and is suitable as a screening test for symptomatic as well as asymptomatic plant material.


2007 ◽  
Vol 70 (9) ◽  
pp. 2015-2022 ◽  
Author(s):  
JOONBAE HONG ◽  
WOO KYUNG JUNG ◽  
JUN MAN KIM ◽  
SO HYUN KIM ◽  
HYE CHEONG KOO ◽  
...  

Campylobacter species are one of the most common causes of bacterial diarrhea in humans worldwide. The consumption of foods contaminated with two Campylobacter species, C. jejuni and C. coli, is usually associated with most of the infections in humans. In this study, a rapid, reliable, and sensitive multiplex real-time quantitative PCR was developed for the simultaneous detection, identification, and quantification of C. jejuni and C. coli. In addition, the developed method was applied to the 50 samples of raw chicken meat collected from retail stores in Korea. C. jejuni and C. coli were detected in 88 and 86% of the samples by real-time quantitative PCR and the conventional microbiological method, respectively. The specificity of the primer and probe sets was confirmed with 30 C. jejuni, 20 C. coli, and 35 strains of other microbial species. C. jejuni and C. coli could be detected with high specificity in less than 4 h, with a detection limit of 1 log CFU/ml by the developed real-time PCR. The average counts (log CFU per milliliter) of C. jejuni or C. coli obtained by the conventional methods and by the real-time PCR assay were statistically correlated with a correlation coefficient (R2) between 0.73 and 0.78. The real-time PCR assay developed in this study is useful for screening for the presence and simultaneous differential quantification of C. jejuni and C. coli.


2019 ◽  
Vol 4 (1) ◽  
pp. 65
Author(s):  
Sandna Larissa Freitas Santos ◽  
Leandro Lima de Vasconcelos ◽  
Rogério Nunes dos Santos

Os intensos períodos de estiagem e pela concentração que a água adquire quando os níveis dos reservatórios estão baixos torna-se evidente a ausência da qualidade da água consumida pela população. O estudo teve como objetivo avaliar os parâmetros físico-químicos e microbiológicos da água para consumo no município de Itapiúna-CE. Estudo do tipo experimental, prospectivo, transversal e com abordagem do tipo qualitativo e quantitativo. Foram analisadas 4 amostras de 500 mL cada, uma do Açude Castro e de três locais escolhidos nas residências de pontos estratégicos de distribuição pela concepcionária, da água usada para consumo no município de Itapiúna-CE no mês de outubro de 2016. Para seleção das amostras foram adotados os critérios de inclusão: água do açude castro (Amostra A), e três amostras de residências, consideradas a água retirada diretamente do registro da Cagece, escolhidas aleatoriamente dos bairros do município. As amostras apresentaram cloretos alterados quando comparadas às quantidades estabelecidas pela Portaria 2914/2011 do Ministério da Saúde (MS). A respeito aos parâmetros como Sólidos Totais, Condutividade, Cloreto e Dureza, os valores também encontrou-se valores superiores. A amostra A (Açude Castro) obteve pH de 8.81 e apresentou alcalinidade devido a carbonatos (80 mgCO3/L) e a bicarbonatos (150mg CaCO3/L). Verificou-se que 50% das amostras apresentaram presença de coliformes totais e coliformes fecais, evidenciando contaminação da maioria das amostras analisadas. Contudo, de acordo com as análises realizadas, as amostras não obedecem ao padrão de potabilidade, tornando á água imprópria para o consumo humano. PHYSICAL-CHEMICAL AND MICROBIOLOGICAL QUALITY OF WATER FOR CONSUMPTION IN RESIDENCES OF A TOWN IN THE SERTÃO CENTRAL ABSTRACT Factors such as long periods of drought and the physical and chemical characteristics of water influence in the quality of water consumed by the population, leading to health problems. This study aimed at evaluating the physical-chemical and microbiological parameters of water for consumption in the town of Itapiúna-CE. Four samples of 500 mL each were analyzed, one from Açude Castro and three from chosen places in the residences of strategic sites of distribution by the water company for consumption in the town of Itapiúna-CE in October 2016. For sample selection it was adopted the inclusion criteria: water from Açude Castro (sample A), and three samples of residences, considered the water withdrawn directly from Cagece register, randomly chosen from neighborhoods of the town. The samples present altered chlorides compared to the established quantities by the Consolidation Ordinance No. 5 of September 28, 2017 of the Ministry of Health (MS). Regarding the parameters such as total solids, conductivity, chloride and hardness, were also found higher values. Sample A (Açude Castro) obtained pH of 8.81 and presented alkalinity due to carbonates (80 mgCO3 / L) and bicarbonates (150 mg CaCO3 / L). It was verified that 50% of the samples presented total coliforms and fecal coliforms, evidencing contamination of most of the samples analyzed. So, according to the analysis carried out, the samples did not obey the standard of potable water, making the water unfit for human consumption.


AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jinghua Ruan ◽  
Wujun Wang ◽  
Tiyin Zhang ◽  
Teng Zheng ◽  
Jing Zheng ◽  
...  

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.


2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Sheng-Ren Sun ◽  
Kashif Ahmad ◽  
Xiao-Bin Wu ◽  
Jian-Sheng Chen ◽  
Hua-Ying Fu ◽  
...  

Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germplasm. In this study, we develop two quick, sensitive, and reliable protocols for real-time quantitative PCR (qPCR) of Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV) using two sets of TaqMan probes and primers targeting the reverse transcriptase/ribonuclease H (RT/RNase H) region. The two assays had a detection limit of 100 copies of plasmid DNA and were 100 times more sensitive than conventional PCR. High specificity of the two assays was observed with respect to SCBIMV and SCBMOV. A total of 176 sugarcane leaf tissue samples from Fujian and Yunnan provinces were collected and analyzed in parallel by conventional PCR, SCBIMV-qPCR, and SCBMOV-qPCR. The SCBIMV-qPCR and SCBMOV-qPCR assays indicated that 50% (88/176) and 47% (83/176) samples tested positive, respectively, whereas only 29% (51/176) tested positive with conventional PCR with the primer pairs SCBV-F and SCBV-R. We demonstrate for the first time that SCBIMV and SCBMOV occur in China and reveal coinfection of both Badnavirus species in 29% (51/176) of tested leaf samples. Our findings supply sensitive and reliable qPCR assays for the detection and quantitation of SCBV in sugarcane quarantine programs.


2004 ◽  
Vol 20 (3) ◽  
pp. 663-664
Author(s):  
James A. Higgins ◽  
Shanavaz Nasarabadi ◽  
Jeffrey S. Karns ◽  
Daniel R. Shelton ◽  
Mary Cooper ◽  
...  

2019 ◽  
Vol 41 ◽  
pp. e55
Author(s):  
Camila Corrêa Bierhas ◽  
Aline Belem Machado ◽  
Simone Ulrich Picoli ◽  
Daniela Montanari Migliavacca Osorio ◽  
Daiane Bolzan Berlese

The contamination of vegetables by pathogenic microorganisms is directly related to the water quality used in their irrigation. Lettuce is the main vegetable consumed in Brazil and because it does not undergo any processing before its consumption, it is directly affected by the quality of the water used for irrigation. This study analyzed the physical-chemical and microbiological quality of water used in lettuce irrigation and possible microbiological contamination of lettuce. In relation to microbiological analyzes, high values were found for heterotrophic bacteria and total coliforms in weirs and vegetables. For fecal coliforms, in almost all water samples, the value found was above that established by the legislation. In lettuce, this occurred in three samples. No strong correlation was found between water and lettuce contamination. In relation to the physicochemical parameters analyzed, only the value of the turbidity in one of the weirs was above the threshold established by the legislation.


2003 ◽  
Vol 18 (9) ◽  
pp. 1115-1123 ◽  
Author(s):  
James A. Higgins ◽  
Shanavaz Nasarabadi ◽  
Jeffrey S. Karns ◽  
Daniel R. Shelton ◽  
Mary Cooper ◽  
...  

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