Simultaneous Detection of Multiple Salmonella Serovars from Milk and Chicken Meat by Real-Time PCR Using Unique Genomic Target Regions

2017 ◽  
Vol 80 (11) ◽  
pp. 1944-1957 ◽  
Author(s):  
Sayma Afroj ◽  
Khaled Aldahami ◽  
Gopal Reddy ◽  
Jean Guard ◽  
Abiodun Adesiyun ◽  
...  
Keyword(s):  
Salmonella Enteritidis ◽  
Simultaneous Detection ◽  
Taqman Assay ◽  
Comparative Genomic ◽  
Pcr Assay ◽  
Salmonella Dublin ◽  
Milk Samples ◽  
Specificity And Sensitivity ◽  
Salmonella Serovars ◽  
Salmonella Heidelberg

ABSTRACT A novel genomic and plasmid target-based PCR platform was developed for the detection of Salmonella serovars Heidelberg, Dublin, Hadar, Kentucky, and Enteritidis. Unique genome loci were obtained through extensive genome mining of protein databases and comparative genomic analysis of these serovars. Assays targeting Salmonella serovars Hadar, Heidelberg, Kentucky, and Dublin had 100% specificity and sensitivity, whereas those for Salmonella Enteritidis had 97% specificity and 88% sensitivity. The limits of detection for Salmonella serovars Heidelberg, Kentucky, Hadar, Enteritidis, and Dublin were 12, 9, 40, 13, and 5,280 CFU, respectively. A sensitivity assay was also performed by using milk artificially inoculated with pooled Salmonella serovars, yielding a detection limit of 1 to10 CFU/25 mL of milk samples after enrichment. The minimum DNA detected using the multiplexed TaqMan assay was 75.8 fg (1.53 × 101 genomic equivalents [GE]) for Salmonella Heidelberg, 140.8 fg (2.8 × 101 GE) for Salmonella Enteritidis, and 3.48 pg (6.96 × 102 GE) for Salmonella Dublin. PCR efficiencies were 89.8% for Salmonella Heidelberg, 94.5% for Salmonella Enteritidis, and 75.5% for Salmonella Dublin. Four types of 30 pasteurized milk samples were tested negative by culture techniques and with a genus-specific Salmonella invA gene PCR assay. Among 30 chicken samples similarly tested, 12 (40%) were positive by both culture and the invA PCR. Testing of these 12 samples with the serovar-specific PCR assay detected single and mixed contamination with Salmonella Kentucky, Salmonella Enteritidis, and Salmonella Heidelberg. Five unique primers were designed and tested by multiplex conventional PCR in conjunction with the use of the multiplex TaqMan assay with three of the primers. The diagnostic assays developed in this study could be used as tools for routine detection of these five Salmonella serovars and for epidemiological investigations of foodborne disease outbreaks.

Real-Time Multiplex SYBR Green I–Based PCR Assay for Simultaneous Detection of Salmonella Serovars and Listeria monocytogenes

2003 ◽  
Vol 66 (11) ◽  
pp. 2141-2145 ◽  
Author(s):  
NARAYANAN JOTHIKUMAR ◽  
XIAOWEN WANG ◽  
MANSEL W. GRIFFITHS
Keyword(s):  
Listeria Monocytogenes ◽  
Salmonella Enteritidis ◽  
Direct Detection ◽  
Simultaneous Detection ◽  
Sybr Green ◽  
Sybr Green I ◽  
Pcr Assay ◽  
Simple Method ◽  
Pcr Products ◽  
Salmonella Serovars

A multiplex SYBR Green I–based PCR assay has been developed for simultaneous detection of Salmonella serovars and Listeria monocytogenes using a LightCycler. Primers were designed to amplify an 85-bp sequence from the gene encoding a fimbrinlike protein (fimI) of Salmonella Enteritidis and a 98-bp sequence from the hemolysin gene (hly) of L. monocytogenes. These primers allowed the amplification of PCR products having distinct melting temperature values, resulting in the formation of two distinct peaks representing the two targets. Background signals, resulting from primer-dimer formation in the late cycles of PCR, are eliminated through the acquisition of data at a high temperature (>75°C), but several degrees lower than required for detection of the specific PCR products. A rapid and simple method for the extraction of bacterial genomic DNA from liquid culture, coupled with duplex PCR using LightCycler SYBR Green–based PCR assays, detected the presence of 2.5 cells and 1 cell of Salmonella serovars and L. monocytogenes, respectively, within an hour. Following overnight enrichment, target DNA was present in sufficient quantities in 1 μl of culture to enable direct detection with the LightCycler.

Prevalence of Salmonella enteritidis and Other Serovars in Ovaries of Layer Hens at Time of Slaughter

1991 ◽  
Vol 54 (7) ◽  
pp. 488-491 ◽  
Author(s):  
HAROLD M. BARNHART ◽  
DAVID W. DREESEN ◽  
ROBERT BASTIEN ◽  
OSCAR C. PANCORBO
Keyword(s):  
Infection Rate ◽  
Salmonella Enteritidis ◽  
Sampling Methods ◽  
Phage Type ◽  
Systemic Infection ◽  
Conventional Methods ◽  
Salmonella Serovars ◽  
Layer Hens ◽  
Salmonella Heidelberg

Ovaries aseptically collected from commercial layer hens at time of slaughter were assayed for Salmonella as an indication of systemic infection of birds within a flock. Birds were randomly selected at the time of slaughter from 42 flocks from seven southeastern states and Pennsylvania. Ovaries were pooled, four per pool, mascerated, and Salmonella, isolates were recovered by conventional methods. Thirty-two of 42 flocks (76.2%) were positive at >10% infection rate based on sampling methods. Fifteen different serovars were detected in flocks. Salmonella heidelberg was the predominant serovar, representing 56.5% of the salmonellae detected. However, S. agona, S. oranienburg, S. mbandaka, S. kentucky, S. montevideo, S. london, S. typhimurium, S. infantis, S. schwarzenqrund, S. ohio, S. cerro, S. anatum, and Salmonella untypeable were also found. S. enteritidis, phage type 23 was recovered from only one (2.4%) of the flocks. Single and multiple serovar infections were found with up to five serovars recovered from a single flock. Twenty-one positive flocks (50%) were positive with a single Salmonella serovar; of these S. heidelberg represented 76.2%. An overall mean of 26.6% of the pooled ovary samples within each infected flock was positive for salmonellae, with an overall range of 0–100%. The significance of Salmonella serovars other than S. enteritidis found at the levels reported has yet to be determined.

Fluorescence in situ hybridization: powerful molecular tool for cancer prognosis

1995 ◽  
Vol 41 (11) ◽  
pp. 1554-1559 ◽  
Author(s):  
J L Fox ◽  
P H Hsu ◽  
M S Legator ◽  
L E Morrison ◽  
S A Seelig
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Simultaneous Detection ◽  
Lymphocytic Leukemia ◽  
Cancer Prognosis ◽  
Comparative Genomic ◽  
Trisomy 8 ◽  
Specificity And Sensitivity ◽  
Genetic Abnormalities

Abstract We review several aspects of fluorescence in situ hybridization (FISH) technology that demonstrate its breadth and power in detecting and monitoring genetic abnormalities associated with cancers. The clinical utility of FISH in disease management is demonstrated in several examples, including trisomy 8 detection with high specificity and sensitivity in patients with myeloid leukemias; trisomy 12 detection with higher efficiency than conventional cytogenetics in patients with chronic lymphocytic leukemia; assessment of engraftment success, chimerism, and relapse in opposite sex bone marrow transplantation; and correlation of trisomy 7 with survival time in patients with prostate tumors. Advances in FISH technology include multicolor analyses, which permit the simultaneous detection of several genetic abnormalities by using cohybridization of probes labeled with several fluorescent labels or label combinations, and comparative genomic hybridization, a relatively new method whereby a single hybridization can reveal aberrations across the entire genome.

Persistence and Growth of Different Salmonella Serovars on Pre- and Postharvest Tomatoes

2007 ◽  
Vol 70 (12) ◽  
pp. 2725-2731 ◽  
Author(s):  
X. SHI ◽  
A. NAMVAR ◽  
M. KOSTRZYNSKA ◽  
R. HORA ◽  
K. WARRINER
Keyword(s):  
Salmonella Enteritidis ◽  
Incubation Temperature ◽  
Narrow Range ◽  
Tomato Fruit ◽  
Diverse Range ◽  
Salmonella Dublin ◽  
Salmonella Newport ◽  
Salmonella Hadar ◽  
Salmonella Serovars ◽  
Salmonella Senftenberg

The interaction of a range of Salmonella serovars with pre- and postharvest tomatoes was evaluated. Serovars were selected on the basis of previous association in tomato-linked outbreaks of salmonellosis (Salmonella Javiana, Salmonella Montevideo, and Salmonella Newport) or those typically isolated from animal or clinical infections (Salmonella Dublin, Salmonella Enteritidis, Salmonella Hadar, Salmonella Infantis, Salmonella Typhimurium, and Salmonella Senftenberg). Salmonella serovars introduced onto the flowers of growing plants were recovered on and within the developing tomato fruit. Of all the Salmonella serovars tested, Montevideo appeared to be more adapted to survival within tomatoes and was recovered from 90% of the fruit screened. All of the Salmonella serovars could persist and grow when introduced onto unripened (green) tomato fruit. In general, growth (internal and external) was promoted at the high incubation temperature (25°C) and high relative humidity (95%), although this was serovar dependent. The growth and persistence of Salmonella introduced on and into ripened (red) tomatoes was serovar dependent. Salmonella serovars Enteritidis, Typhimurium, and Dublin were less adapted to grow in or on intact red tomatoes than were serovars Hadar, Montevideo, or Newport. The results illustrated that a diverse range of Salmonella serovars can become established within and/or on preharvest tomatoes. The majority of Salmonella can grow and become established both on and within unripened tomatoes, but growth on ripened fruit was serovar dependent. The results provide a possible explanation why only a narrow range of Salmonella serovars are associated with foodborne illness outbreaks linked to tomatoes.

Real-Time PCR Method for Quantification of Staphylococcus aureus in Milk

2007 ◽  
Vol 70 (1) ◽  
pp. 90-96 ◽  
Author(s):  
M. GOTO ◽  
H. TAKAHASHI ◽  
Y. SEGAWA ◽  
H. HAYASHIDANI ◽  
K. TAKATORI ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
High Specificity ◽  
Pcr Assay ◽  
Milk Samples ◽  
High Temperature Short Time ◽  
Specificity And Sensitivity ◽  
Pcr Method

A reproducible real-time PCR method that targets the putative transcriptional regulator gene of Staphylococcus aureus was developed to quantify this microorganism in milk samples. On the basis of partial sequences of this gene determined from S. aureus strains, we designed the specific primers and probe for use in a quantitative PCR assay. These specificities were confirmed with 25 strains of S. aureus and 35 strains of other bacteria. A real-time PCR assay with serial 10-fold dilutions of purified DNA and pure culture was conducted. It was possible to construct standard curves with a high correlation coefficient (r2 = 0.99) in the range of 50 ng to 50 fg for purified DNA and 107 to 101 CFU/ml for a pure culture. The constructed standard curve for milk samples was similar to that for the pure culture, and the quantification of S. aureus in the range of 107 to 101 CFU/ml was possible. Moreover, to determine how our real-time PCR method would perform under actual analytical conditions, we quantified the DNA from S. aureus after two types of heat treatments were used for the pasteurization of milk. The amount of DNA found was affected after heat treatment at 63°C for 30 min (low-temperature long-time method) but not at 72°C for 15 s (high-temperature short-time method). The results indicate that the real-time PCR method developed in this study is effective for monitoring S. aureus contamination in milk because of its high specificity and sensitivity.

scholarly journals Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

2014 ◽  
Vol 53 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Piyumali K. Perera ◽  
Robin B. Gasser ◽  
Simon M. Firestone ◽  
Lee Smith ◽  
Florian Roeber ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Simultaneous Detection ◽  
Asia Pacific ◽  
Analytical Sensitivity ◽  
Pacific Region ◽  
Asia Pacific Region ◽  
Pcr Assay ◽  
Theileria Orientalis ◽  
Content Type ◽  
Specificity And Sensitivity

Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of theTheileria orientaliscomplex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of theT. orientaliscomplex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of theT. orientaliscomplex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes ofT. orientaliscomplex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region.

scholarly journals A Novel Multiplex RT-PCR Assay for Simultaneous Detection of Dengue and Chikungunya Viruses

2020 ◽  
Vol 21 (21) ◽  
pp. 8281
Author(s):  
Mohammed Alimul Islam ◽  
Mohamed E. El Zowalaty ◽  
Sumaiya Islam ◽  
Mohiuddin Sharif ◽  
Md. Rajibur Rahman ◽  
...  
Keyword(s):  
Simultaneous Detection ◽  
Cross Reactivity ◽  
Single Step ◽  
Epidemiological Surveillance ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Serum Samples ◽  
Viral Genomes ◽  
Specificity And Sensitivity ◽  
Field Samples

The goal of the study was to develop a specific, sensitive, and cost-effective molecular RT-PCR diagnostic assay for the rapid and simultaneous detection of the serotypes of dengue virus (DENV) and Chikungunya virus (CHIKV) from sera of suspected febrile patients. A single-tube, single-step multiplex RT-PCR (mRT-PCR) assay was designed for the detection of viral genomes from clinical and field samples. Specificity and sensitivity of the mRT-PCR assay were evaluated against six different combinations using two reverse transcriptases (AMV-RT and RT-Ace) and three DNA polymerases (LA-Taq, rTaq, and Tth). Among the six combinations, the AMV-RT and LA-Taq combination was more specific and sensitive than other enzyme combinations for detecting viral genomes of DENV-1, DENV-2, DENV-3, and DENV-4 (p < 0.01), and for detecting viral genomes of CHIKV (p < 0.05). The detection limits of the mRT-PCR were 10 focus forming units (FFU) for CHIKV and 1 FFU, 20 FFU, 0.1 FFU, and 10 FFU for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. The primers used for the mRT-PCR did not show any cross-reactivity among the serotypes of DENV or CHIKV. Specificity and sensitivity of the newly developed mRT-PCR were validated using serum samples collected from febrile patients during dengue outbreaks in Bangladesh. The sensitivity for serotype detection of DENV and CHIKV was superior to the virus isolation method and the antigen detection method using the Dengue NS1-Ag assay. This novel mRT-PCR method can be used for molecular epidemiological surveillance of DENV and CHIKV in epidemic and endemic countries.

scholarly journals Duplex real-time PCR assay for the simultaneous detection of Achromobacter xylosoxidans and Achromobacter spp

2020 ◽  
Author(s):  
Erin P. Price ◽  
Valentina Soler Arango ◽  
Timothy J. Kidd ◽  
Tamieka A. Fraser ◽  
Thuy-Khanh Nguyen ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Diagnostic Methods ◽  
Achromobacter Xylosoxidans ◽  
Resistant Bacteria ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Pathogenic Potential ◽  
Specificity And Sensitivity

AbstractSeveral members of the Gram-negative environmental bacterial genus, Achromobacter, are associated with serious infections in immunocompromised individuals, of which Achromobacter xylosoxidans is the most common. Despite their pathogenic potential, comparatively little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management of Achromobacter infections. Here, we performed comparative genomics of 158 Achromobacter spp. genomes to robustly identify species boundaries, to reassign several incorrectly speciated taxa, and to identify genetic sequences specific for the Achromobacter genus and for A. xylosoxidans. Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of Achromobacter spp. and A. xylosoxidans from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as Achromobacter spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of Achromobacter spp. and A. xylosoxidans, with five Achromobacter isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both Achromobacter spp. and A. xylosoxidans down to ∼110 genome equivalents, and detected down to ∼12 and ∼1 genome equivalent/s, respectively. In silico analysis, and laboratory testing of 34 non-Achromobacter isolates and 38 adult CF sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive, and cost-effective method for the simultaneous detection of all Achromobacter spp. and A. xylosoxidans, and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

Sign in / Sign up

Export Citation Format

Share Document