Multiplex-direct PCR assay for foodborne pathogen identification: An application in forensic investigation

2013 ◽  
Vol 4 (1) ◽  
pp. e103-e104 ◽  
Author(s):  
Laila Hasap ◽  
Phuvadol Thanakiatkrai ◽  
Kamonnut Singkhamanan ◽  
Adrian Linacre ◽  
Thitika Kitpipit
Keyword(s):  
Foodborne Pathogen ◽  
Forensic Investigation ◽  
Pathogen Identification ◽  
Pcr Assay ◽  
Direct Pcr

scholarly journals Occurrence, antimicrobial susceptibility patterns and genotypic relatedness of Salmonella spp. isolates from captive wildlife, their caretakers, feed and water in India

2018 ◽  
Vol 146 (12) ◽  
pp. 1543-1549
Author(s):  
Arockiasamy Arun Prince Milton ◽  
Rajesh Kumar Agarwal ◽  
Govindarajan Bhuvana Priya ◽  
Cheruplackal Karunakaran Athira ◽  
Mani Saminathan ◽  
...  
Keyword(s):  
Antimicrobial Susceptibility ◽  
Animal Species ◽  
Multidrug Resistant ◽  
Wild Animal ◽  
Diffusion Method ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Salmonella Spp ◽  
Culture Methods ◽  
Faecal Samples

AbstractOccurrence of Salmonella spp. in captive wild animal species in India is largely unknown. The purpose of this study was to determine the occurrence of different Salmonella serotypes, antimicrobial resistance patterns and genotypic relatedness of recovered isolates. A total of 370 samples including faecal (n = 314), feed and water (n = 26) and caretakers stool swabs (n = 30) were collected from 40 different wild animal species in captivity, their caretakers, feed and water in four zoological gardens and wildlife enclosures in India. Salmonellae were isolated using conventional culture methods and tested for antimicrobial susceptibility with the Kirby–Bauer disc diffusion method. Salmonella isolates were serotyped and genotyping was performed using enterobacterial repetitive intergenic consensus (ERIC) PCR and 16S rRNA sequencing. Animal faecal samples were also subjected to direct PCR assay. Salmonella was detected in 10 of 314 (3.1%) faecal samples by isolation and 18 of 314 (5.7%) samples by direct PCR assay; one of 26 (3.8%) feed and water samples and five of 30 (16.7%) caretakers stool swabs by isolation. Salmonella was more commonly isolated in faecal samples from golden pheasants (25%; 2/8) and leopard (10%; 2/20). Salmonella enterica serotypes of known public health significance including S. Typhimurium (37.5%; 6/14), S. Kentucky (28.5%; 4/14) and S. Enteritidis (14.3%; 2/14) were identified. While the majority of the Salmonella isolates were pan-susceptible to the commonly used antibiotics. Seven (43.7%; 7/16) of the isolates were resistant to at least one antibiotic and one isolate each among them exhibited penta and tetra multidrug-resistant types. Three S. Kentucky serotype were identified in a same golden pheasants cage, two from the birds and one from the feed. This serotype was also isolated from its caretaker. Similarly, one isolate each of S. Typhimurium were recovered from ostrich and its caretaker. These isolates were found to be clonally related suggesting that wildlife may serve as reservoir for infections to humans and vice versa. These results emphasise the transmission of Salmonella among hosts via environmental contamination of feces to workers, visitors and other wildlife.

scholarly journals First Report of Flavescence Dorée-Related Phytoplasma Affecting Grapevines in Croatia

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 353-353 ◽  
Author(s):  
M. Šeruga Musić ◽  
D. Škorić ◽  
I. Haluška ◽  
I. Križanac ◽  
J. Plavec ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Reference Strain ◽  
Simultaneous Detection ◽  
Pinot Noir ◽  
Vitis Vinifera L ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Flavescence Dorée ◽  
Scaphoideus Titanus

Flavescence dorée (FD) and Bois noir (BN) phytoplasmas are principal grapevine yellows (GY) agents in the wider Euro-Mediterranean Region. While BN phytoplasma belongs to the ribosomal subgroup 16SrXII-A, the FD agents belong either to the ribosomal subgroups 16SrV-C or -D. During the official GY survey in 2009, 40 symptomatic grapevines (Vitis vinifera L.) were sampled throughout grapevine-growing regions in Croatia. Typical GY symptoms of leaf yellowing or reddening were evident on white and red varieties, respectively. Leaf rolling as well as irregular lignification of the shoots and withering of clusters were also observed. Phloem tissue from cuttings and leaf veins from mature vines were sampled for total DNA extraction and amplification of phytoplasma 16S rRNA gene by using generic primers P1/P7 in a direct PCR assay followed by a nested PCR using primer pair R16F2n/R2 (2). Phytoplasma ribosomal group affiliation was determined by restriction fragment length polymorphism (RFLP) analysis of the nested PCR products with enzyme Tru1I (Fermentas, Vilnius, Lithuania). These initial findings were validated and augmented by a triplex real-time PCR assay targeting the nonribosomal map gene. This assay enables simultaneous detection of BN and FD (16SrV-C and -D) phytoplasmas in grapevine (3). Assay results revealed the majority of GY positive vines (19 of 40) contained BN phytoplasma which is widespread. For the first time in Croatia, two red variety samples, Pinot Noir and Plemenka Crvena, from the vicinity of Ozalj (Vivodina) and Zagreb (Brezje), respectively, were found to harbor FD-related phytoplasmas. Fragments amplified by P1/P7 primers from latter samples were cloned and sequenced. Sequence analyses using online interactive tool iPhyClassifier (4) revealed that the phytoplasma under study from Pinot Noir sample (GenBank Accession No. HQ712064) is a member of 16SrV-C subgroup and shares 99.87% similarity with 16S rDNA sequence of the reference strain (GenBank Accession No. AF176319). The sequence from the Plemenka Crvena sample (GenBank Accession No. HQ712065) shares 99.54% similarity with the reference strain and has the most similar virtual RFLP pattern to the one of the 16SrV-C subgroup (GenBank Accession No. AY197642). These findings are currently limited to vineyards in northwestern Croatia. Even so, the presence of FD principal cicadellid vector Scaphoideus titanus in the country and the occurrence and distribution of FD in neighboring countries (1,2) are factors indicating that the spread of FD in Croatia is highly probable. References: (1) L. Filippin et al. Plant Pathol. 58:826, 2009. (2) S. Kuzmanović et al. Vitis 47:105, 2008. (3) C. Pelletier et al. Vitis 48:87, 2009. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

Are these food products fraudulent? Rapid and novel triplex-direct PCR assay for meat identification

2013 ◽  
Vol 4 (1) ◽  
pp. e33-e34 ◽  
Author(s):  
Thitika Kitpipit ◽  
Kuangtiwa Sittichan ◽  
Phuvadol Thanakiatkrai
Keyword(s):  
Food Products ◽  
Pcr Assay ◽  
Direct Pcr

scholarly journals SalivaSTAT: Direct-PCR and pooling of saliva samples collected in healthcare and community setting for SARS-CoV-2 mass surveillance

2020 ◽  
Author(s):  
Nikhil S Sahajpal ◽  
Ashis K Mondal ◽  
Sudha Ananth ◽  
Allan Njau ◽  
Pankaj Ahluwali ◽  
...  
Keyword(s):  
Clinical Sample ◽  
Diagnostic Testing ◽  
Rna Extraction ◽  
Community Setting ◽  
Absolute Quantification ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Sample Pooling ◽  
Saliva Collection

BackgroundThe limitations of widespread current COVID-19 diagnostic testing lie at both pre-analytical and analytical stages. Collection of nasopharyngeal swabs is invasive and is associated with exposure risk, high cost, and supply-chain constraints. Additionally, the RNA extraction in the analytical stage is the most significant rate-limiting step in the entire testing process. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction free RT-PCR test using any of the commercially available RT-PCR kits.MethodsWe optimized saliva collection devices, heat-shock treatment and homogenization. The effect of homogenization on saliva samples for extraction-free RT-PCR assay was determined by evaluating samples with and without homogenization and preforming viscosity measurements. Saliva samples (872) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. Further, a five-sample pooling strategy was evaluated as per FDA guidelines using the SalivaSTAT protocol.ResultsThe saliva collection (done without any media) performed comparable to the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95°C for 30-minutes and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreement (NPA) of 95.8% and 100%, respectively. The LoD was established as ∼20-60 copies/ml by absolute quantification. Further, a five-sample pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively.ConclusionWe have optimized an extraction-free direct RT-PCR assay for saliva samples that demonstrated comparable performance to FDA-EUA assay (Extraction and RT-PCR). The SalivaSTAT protocol is a rapid, sensitive, and cost-effective method that can be adopted globally, and has the potential to meet testing needs and may play a significant role in management of the current pandemic.

Detection of Campylobacter from Poultry Carcass Skin Samples at Slaughter in Southern Italy

2009 ◽  
Vol 72 (8) ◽  
pp. 1718-1721 ◽  
Author(s):  
TIZIANA PEPE ◽  
ROSARIA DE DOMINICIS ◽  
GIUSEPPINA ESPOSITO ◽  
IOLE VENTRONE ◽  
PINA M. FRATAMICO ◽  
...  
Keyword(s):  
Southern Italy ◽  
Control Strategies ◽  
Foodborne Pathogen ◽  
Raw Milk ◽  
Human Infection ◽  
Contaminated Water ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Human Infections ◽  
Poultry Carcass

Campylobacter is a major foodborne pathogen responsible for acute gastroenteritis characterized by diarrhea that is sometimes bloody, fever, cramps, and vomiting. Campylobacter species are carried in the intestinal tracts of mammals and birds, and sources of human infection include raw milk, contaminated water, direct contact with pets, and foods, particularly poultry. Campylobacter jejuni and C. coli are the species that account for the majority of human infections. The aim of this work was to determine the prevalence of Campylobacter in 190 poultry carcasses sampled at slaughter and to use a multiplex PCR assay to determine if the isolates were C. jejuni or C. coli. C. coli was not isolated, while C. jejuni was recovered from 52 (37.1%) of 140 carcasses for which pools of four sampling sites (neck, cloaca, breast, and back) were examined. In the remaining 50 carcasses, the four sites were analyzed separately, and C. jejuni was recovered from the samples in the following order: neck (n = 20), cloaca (n = 16), breast (n = 14), and back (n = 11). The results are in agreement with those of other studies, which showed that C. jejuni is more commonly associated with poultry than is C. coli. Control strategies for Campylobacter should include interventions to eliminate C. jejuni in poultry at various stages of production and processing, including at slaughter.

scholarly journals SalivaSTAT: Direct-PCR and Pooling of Saliva Samples Collected in Healthcare and Community Setting for SARS-CoV-2 Mass Surveillance

Diagnostics ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 904
Author(s):  
Nikhil S. Sahajpal ◽  
Ashis K. Mondal ◽  
Sudha Ananth ◽  
Allan Njau ◽  
Pankaj Ahluwalia ◽  
...  
Keyword(s):  
Clinical Sample ◽  
Diagnostic Testing ◽  
Community Setting ◽  
Absolute Quantification ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Comparable Performance ◽  
Sample Pooling ◽  
Saliva Collection

Objectives: Limitations of widespread current COVID-19 diagnostic testing exist in both the pre-analytical and analytical stages. To alleviate these limitations, we developed a universal saliva processing protocol (SalivaSTAT) that would enable an extraction-free RT-PCR test using commercially available RT-PCR kits. Methods: We optimized saliva collection devices, heat-shock treatment, and homogenization. Saliva samples (879) previously tested using the FDA-EUA method were reevaluated with the optimized SalivaSTAT protocol using two widely available commercial RT-PCR kits. A five-sample pooling strategy was evaluated as per FDA guidelines. Results: Saliva collection (done without any media) showed performance comparable to that of the FDA-EUA method. The SalivaSTAT protocol was optimized by incubating saliva samples at 95 °C for 30-min and homogenization, followed by RT-PCR assay. The clinical sample evaluation of 630 saliva samples using the SalivaSTAT protocol with PerkinElmer (600-samples) and CDC (30-samples) RT-PCR assay achieved positive (PPA) and negative percent agreements (NPAs) of 95.0% and 100%, respectively. The LoD was established as ~60–180 copies/mL by absolute quantification. Furthermore, a five-sample-pooling evaluation using 250 saliva samples achieved a PPA and NPA of 92% and 100%, respectively. Conclusion: We have optimized an extraction-free RT-PCR assay for saliva samples that demonstrates comparable performance to FDA-EUA assay (Extraction and RT-PCR).

Detection of Escherichia albertii in retail oysters

2021 ◽  
Author(s):  
Sakura Arai ◽  
Satoko Yamaya ◽  
Kayoko Ohtsuka ◽  
Noriko Konishi ◽  
Hiromi Obata ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Pacific Oyster ◽  
Genetic Characterization ◽  
Foodborne Pathogen ◽  
Most Probable Number ◽  
Macconkey Agar ◽  
Pcr Assay ◽  
Probable Number ◽  
Pacific Oysters ◽  
Rock Oyster

Escherichia albertii  is an emerging foodborne pathogen. Owing to its distribution in river water,  it is important to determine the presence of  E. albertii  in aquaculture-related foods. In this study, we investigated the distribution of  E. albertii  in retail oyster samples.  A total of  427 raw oyster samples (385 Pacific oysters, and 42 Japanese rock oysters) were enriched in  modified Escherichia coli  broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42 °C. The cultures were used for  E. albertii -specific nested PCR assay, as well as for  E. albertii  isolation using  deoxycholate hydrogen sulfide lactose agar  (DHL), DHL supplemented with rhamnose and xylose (RX-DHL), and MacConkey agar supplemented with rhamnose and xylose (RX-MAC). The population of  E. albertii  in nested PCR-positive samples was  determined using the  most probable number  (MPN) method.  E. albertii  isolates were subjected to biochemical and genetic characterization.  E. albertii   was detected in 5 of 315 (1.6%) Pacific oyster samples  (one piece each), 2 of 70 (2.9 %)  Pacific oyster samples  (25 g each), and 2 of 42 (4.8 %) Japanese rock oyster samples  procured from four geographically distant regions. A total of 64  E. albertii  strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and  the MPN value was under the detection limit (< 3 MPN/10 g).  A specific season or month for detecting  E. albertii  was not observed in this study, suggesting that the pathogen is present in seawater.   All the  E. albertii  isolates, except one, were positive for the virulence factor  eae,  indicating that these isolates have  the potential to infect humans.

Direct Detection and Identification of Arcobacter Species by Multiplex PCR in Chicken and Wastewater Samples from Spain

2007 ◽  
Vol 70 (2) ◽  
pp. 341-347 ◽  
Author(s):  
A. GONZÁLEZ ◽  
S. BOTELLA ◽  
R. M. MONTES ◽  
Y. MORENO ◽  
M. A. FERRÚS
Keyword(s):  
Multiplex Pcr ◽  
Water Samples ◽  
Direct Detection ◽  
Pcr Amplification ◽  
Pcr Assay ◽  
Direct Pcr ◽  
Culture Methods ◽  
Multiplex Pcr Assay ◽  
Detection And Identification ◽  
Wastewater Samples

Twenty-two chicken livers, 10 chicken carcasses, and 15 wastewater samples were processed and analyzed for Arcobacter by PCR and traditional culture methods. Samples were enriched for 24 and 48 h, incubated at 30°C under aerobic conditions, and streaked on blood selective media. To determine the best isolation conditions, 20 samples also were processed under microaerophilic conditions at 37°C. Simple and multiplex PCR assays were used directly with enrichment broths and isolated strains. Seventeen Arcobacter strains were isolated from chicken samples, and A. butzleri was the only Arcobacter species identified. The direct PCR assay revealed that 29 of the 32 chicken samples were contaminated with Arcobacter. A. butzleri was the most frequently detected species, although Arcobacter cryaerophilus also was present in some of the samples and Arcobacter skirrowii occasionally was detected. All the wastewater samples were positive by PCR assay for Arcobacter after 24 h of enrichment. A. butzleri and A. cryaerophilus were detected with the multiplex PCR assay. Fourteen Arcobacter strains were isolated from 10 of the 15 water samples analyzed; 7 were identified as A. butzleri and the remaining 7 were A. cryaerophilus. Both for chicken and water samples, Arcobacter detection rate for PCR amplification was higher than for culture isolation. These results indicate the high prevalence of Arcobacter in chicken and wastewater and the inadequacy of available cultural methods for its detection. The species-specific multiplex PCR assay is a rapid method for assessing Arcobacter contamination in chicken and wastewater samples and is a viable alternative to biochemical identification of isolated strains.

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