Simplex Polymerase Chain Reaction Research Articles

A Duplex PCR Assay for Rapid Detection of Klebsiella pneumoniae and Chryseobacterium in Large Yellow Croaker Fish.

Both Klebsiella pneumoniae and Chryseobacterium cause an increasing number of diseases in fish, resulting in great economic losses in aquaculture. In addition, the disease infected with Klebsiella pneumoniae or Chryseobacterium exhibited the similar clinical symptoms in aquatic animals. However, there is no effective means for the simultaneous detection of co-infection and discrimination them for these two pathogens. Here, we developed a duplex polymerase chain reaction (PCR) method based on the outer membrane protein A (ompA) gene of Klebsiella pneumoniae and Chryseobacterium. The specificity and validity of the designed primers were confirmed experimentally using simplex PCR. The expected amplicons for Klebsiella pneumoniae and Chryseobacterium had a size of 663 and 1404 bp, respectively. The optimal condition for duplex PCR were determined to encompass a primer concentration of 0.5 μM and annealing temperature of 57°C. This method was analytical specific with no amplification being observed from the genomic DNA of Escherichia coli, Vibrio harveyi, Pseudomonas plecoglossicida, Aeromonas hydrophila and Acinetobacter johnsonii. The limit of detection was estimated to be 20 fg of genomic DNA for Chryseobacterium and 200 fg for Klebsiella pneumoniae, or 100 colony-forming units (CFU) of bacterial cells in both cases. The duplex PCR was capable of simultaneously amplifying target fragments from genomic DNA extracted from the bacteria and fish liver. For practical validation of the method, 20 diseased fish were collected from farms, among which 4 samples were PCR-positive for Klebsiella pneumoniae and Chryseobacterium. The duplex PCR method developed here is time-saving, specific, convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of Klebsiella pneumoniae and Chryseobacterium in the field of aquaculture.

Validating duplex-PCR targeting ND2 for bovine and porcine detection in meat products

Food authentication is a mandatory effort to assure the fair-trade. This study developed a duplex polymerase chain reaction (PCR) from the NADH dehydrogenase subunit 2 (ND2) gene to amplify specific segments of a cattle and porcine DNA. A universal forward primer composed of nineteen base pairs (bp) (3′-CCAAACACAACTCCGAAAA-5′) and species-specific reverse primers composed of twenty (3′-CCAAACACAACTCCGAAAA-5′) and twenty-one (3′-TGGCAAGAATTAGGACGGTTA-5′) bp were used to limit the amplified DNA segment for porcine and cattle. The PCR reaction would generate a product with a profile of 168 and 227 bp, respectively. To investigate the accuracy and limit of detection, an in vitro experiment was conducted using simplex and duplex PCR on commercial meatballs randomly purchased from a commercial market in Surakarta, Indonesia. The findings of this study indicated that ND2 could be used as an alternative genetic marker for the identification of porcine and beef species in meat-derived products.

Polymerase chain reaction-based snake origin tracing in commercial venom crystals by targeting the mitochondrial D-loop

Snake venom is a valuable raw material for numerous therapeutic formulations because of its life-saving pharmacological potential. However, due to their high price, fake “snake venoms” have captured a significant portion of the global market, and there is currently no reliable reported DNA-based method available for quickly distinguishing between fakes and originals. Therefore, in this study, a set of newly designed snake-specific universal primers targeting mitochondrial D-loop fragments were employed to detect snake origins in commercial venom crystals by only simplex polymerase chain reaction analysis. Under the optimal thermal cycling conditions, only the 145–149 bp snake-specific mitochondrial D-loop fragments from pure and mixed backgrounds were amplified by the newly designed primers. Specificity was achieved by confirming no DNA amplification occurred in the DNA admixture of ten different chordates, and universality by individual DNA amplification of nine different snakes. The primers that efficiently amplified the minimum mitochondrial DNA contained in a total of 10−2 ng in a 10.0 μl reaction were also successfully able to detect the snake origin in commercial cobra venom crystals. These findings suggest that the newly designed primers can be used to differentiate the original and fake commercial snake venom crystals in order to achieve the highest standards of snake venom-based medications through amplifying the snake-specific mitochondrial D-loop fragments.

Serogroups and genetic diversity of diarrheagenic strains of Escherichia coli: a retrospective study.

Diverse serogroups of Escherichia coli cause sporadic cases and outbreaks of diarrhea among children. Our study aimed to evaluate the serogroups of diarrheagenic strains of E. coli that cause diarrheal disease in children under two years old, and clarify if the cases were sporadic or outbreaks. The retrospective study included 130 strains of pathogenic E. coli, isolated from children who were less than two years of age, and had diarrheal disease, between May 2016 and July 2019. The study was conducted in the Bacteriology Laboratory (County Clinical Hospital, Mureș, Romania). The 130 strains were sero-grouped using polyvalent and monovalent O antisera. Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR) was performed to evaluate the similarity between different E. coli strains, and a simplex polymerase chain reaction (PCR) was performed to detect the presence of the hlyA gene that is specific to the enterohemorrhagic strains. After agglutination with polyvalent O antisera, slightly more than half of the strains (50.77%) were sero-grouped as Shiga toxin-producing E. coli (STEC), and the rest of the strains belonged to the Enteropathogenic Escherichia coli (EPEC) serogroups. Serogroup O157 was the most frequently identified (16.51% of the total number of typeable strains), and one strain was positive for hlyA. ERIC-PCR revealed a high diversity of strains, with an overall 50% similarity. STEC serogroups were the most common strains causing diarrheal disease, and O-157 was the dominant serogroup identified. The strains included in our study presented high genetic diversity, suggesting that most of the cases were sporadic.

Targeted (PCR-based) screening of antibiotic resistance genes' prevalence in the gut microbiota of tribal people of Nabarangpur, Odisha, India.

Antibiotic resistance is a major public health concern worldwide. The gut microbiota harbours multiple antibiotic resistant genes (ARGs) that contribute to the existing and future microbial population in a community or ecosystem. This study aimed to investigate the prevalence of 35 antibiotic resistance genes (ARGs) in the gut microbiota of the tribal people of Nabarangpur, Odisha, India. A total of 83 faecal samples were collected from three different tribes (Bhatra, Gond, and Paraja). Total faecal DNA was extracted, and the simplex polymerase chain reaction was performed to detect selected ARGs. Further analysis was done to estimate the incidence of these ARGs across these tribes based on alcohol consumption habits. We identified a higher prevalence of tetracycline resistance genes (tetW, tetQ and tetM) in the gut microbiota among three populations. Furthermore, a significant (P = 0·024) difference in ARG prevalence against vancomycin in individuals with and without alcohol consumption habits was noticed. The overall distribution of ARGs among the three major tribes of this location was found to be very similar. Together, irrespective of the tribes, the people of this location have gut microbiota harbouring different kinds of ARGs and tetracycline-resistant genes are the most commonly found ARGs.

Detection and Molecular Identification of Eight Candida Species in Clinical Samples by Simplex PCR.

Systemic candidiasis is a frequent opportunistic mycosis that can be life-threatening. Its main etiological agent is Candida albicans; however, the isolation of non-albicans Candida species has been increasing. Some of these species exhibit greater resistance to antifungals, so the rapid and specific identification of yeasts is crucial for a timely diagnosis and optimal treatment of patients. Multiple molecular assays have been developed, based mainly on polymerase chain reaction (PCR), showing high specificity and sensitivity to detect and identify Candida spp. Nevertheless, its application in diagnosis has been limited due to specialized infrastructure or methodological complexity. The objective of this study was to develop a PCR assay that detects and identifies some of the most common pathogenic Candida species and evaluate their diagnostic utility in blood samples and bronchial lavage. A pair of oligonucleotides was designed, CandF and CandR, based on sequence analysis of the 18S-ITS1-5.8S-ITS2-28S region of the rDNA of Candida spp., deposited in GenBank. The designed oligonucleotides identified C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei/Pichia kudriazevii, C. guilliermondii/Meyerozyma guilliermondii, C. lusitaniae/Clavispora lusitaniae, and C. dubliniensis using simplex PCR based on the amplicon size, showing a detection limit of 10 pg/μL of DNA or 103 yeasts/mL. Based on cultures as the gold standard, it was determined that the sensitivity (73.9%), specificity (96.3%), and the positive (94.4%) and negative (81.2%) predictive values of the PCR assay with the designed oligonucleotides justify their reliable use in diagnosis.

A multiplex polymerase chain reaction for the simultaneous detection of the virus and satellite components associated with cotton leaf curl begomovirus disease complex

Cotton leaf curl disease (CLCuD) is caused by a complex of several whiteflies (Bemisia tabaci Genn.)-transmitted begomovirus species, Cotton leaf curl Multan virus (CLCuMuV), Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Alabad virus (CLCuAlV) by individual of mixed infection, associated with Cotton leaf curl Multan betasatellite (CLCuMB) and several alphasatellites. The disease causes major economic losses in cotton in the Indian subcontinent. For monitoring of epidemiology and development of management strategies of CLCuD, a quick, sensitive and effective method capable of detecting all the begomovirus, betasatellite and alphasatellite components associated with CLCuD is required. With this objective, a multiplex polymerase chain reaction (mPCR) assay was developed for the simultaneous detection of these three viral components associated with CLCuD of cotton. Primers for each component were designed based on the retrieved reference sequences from the GenBank. Each pair of primers, designed for each of the respective component, was evaluated for its sensitivity and specificity in both the component-specific simplex polymerase chain reaction (sPCR) and mPCR assay. This report identified three viral component-specific pairs of primers which, in all combinations, amplified simultaneously the CP gene (780 nts) of the begomovirus, the βC1gene (375 nts) of the betasatellite and the Rep gene (452 nts) of the alphasatellite associated with CLCuD in the mPCR assays. The amplified products specific to each component produced by these assays were identified based on their amplicon sizes, and the identities of the viral components amplified were confirmed by cloning and sequencing the amplicons obtained in the mPCR. The mPCR assay was validated using naturally CLCuD-affected cotton plants of the fields. This assay will be useful for rapid detection of CLCuD-associated begomovirus, betasatellite and alphasatellite DNA in field samples, extensive resistance screening in resistance breeding programme, and also monitoring epidemiology for detection of virus and its components when symptoms are mild or absent in the plant.

Investigation of antiseptic resistance genes in Staphylococcus spp. isolates

Antiseptic and disinfectants are used very frequently in all health institutions, including hospital and veterinary application areas, in the home environment and food production industry, to prevent infections and contaminations. At present, the quaternary ammonium compounds, benzalkonium chloride and chlorhexidine digluconate is one of the divalent cations most commonly used chemicals such as antiseptics and disinfectants. However, the widespread use of biocides has brought about the emergence of bacteria resistant to antiseptics/disinfectants. It is known that bacteria develop resistance mechanisms against antibiotics as well as disinfectants. Epidemiological data on antiseptic susceptibility and distribution of resistance genes are very important for nosocomial infections. Some species, including the species belong to genus Staphylococcus, cause foodborne poisoning and various clinical infections such as skin and soft tissue and surgical site infections, endocarditis, mastitis, pneumonia and bacteremia in humans and animals. Staphylococcus strains can contain plasmid-derived qacA/B and qacC genes that provide resistance to quaternary ammonium compounds (QAC). In this study, the presence of antiseptic resistance genes (qacA/B and qacC) in 90 Staphylococcus spp. strains isolated from chicken carcass, bovine tank milk, various cheeses and bovine clinical mastitis samples were determined by simplex polymerase chain reaction. QacA/B was found in %18.8 and qacC in %2.2 of the studied isolates. Of antiseptic resistance genes, qacA/B was detected in cheese and bovine clinical mastitis samples, and qacC in chicken carcass.

Antimicrobial Resistance Determinant of Vibrio species recovered from selected freshwaters in Southwest Nigeria

Antimicrobial Resistance Determinant of Vibrio species recovered from selected freshwaters in South West Nigeria1Ibukun Modupe Adesiyan and 1Mary Adejumoke Bisi-Johnson,21Institute of Ecology and Environmental Studies, Obafemi Awolowo University, Ile-Ife, Nigeria 2Department of Microbiology, Obafemi Awolowo University, Ile-Ife, NigeriaBACKGROUND: Reduction in the burden of infectious diseases has been made possible since the discovery of antimicrobial agent. However, the development of resistance and its quick spread is negating the effectiveness of antibiotics therapy. METHODS: A total of 315 Vibrio isolates recovered from selected freshwater sources in South West Nigeria were confirmed by simplex polymerase chain reaction technique using toxR gene. All confirmed isolates were tested for In vitro susceptibility to 18 antibiotics using the standardized agar disc-diffusion assay and the resistant isolates were further profiled for their genotypic antimicrobial resistance determinants by PCR. RESULTS: Of the 18 antimicrobials selected from 10 families, resistance among sulphonamides (94%), Macrolides (95%), and tetracyclines (83%), occurred with higher frequency than others. Multi-drug resistance ranged from three to nine antimicrobials. Prevalence and distributions of 11 resistance determinant were obtained as follows; [sulfonamides (sulI (19%), sulII (33%)], [beta-lactams; (ampC 39%; blaOXA (27%), blapse (11%)], and [aminoglycosides; (aacC2 (24%), aphA1 (14%),strA (39%)]. Strong positive correlations between Cd and blapse, Cr and sulII (Vibrio spp.) and As and aphA2 (P. shigelloides) were observed. CONCLUSION: This study revealed high prevalence of multidrug resistance Vibrio spp. and their resistance determinant in this environment. This indicates that these fresh waters poses health risk to the populace if consumed untreated. Therefore there is need for continuous surveillance and monitoring of water bodies in order to safeguard the health of the populace.

A rapid and accurate method for the detection of four aminoglycoside modifying enzyme drug resistance gene in clinical strains of Escherichia coli by a multiplex polymerase chain reaction.

BackgroundAntibiotics are highly effective drugs used in the treatment of infectious diseases. Aminoglycoside antibiotics are one of the most common antibiotics in the treatment of bacterial infections. However, the development of drug resistance against those medicines is becoming a serious concern.AimThis study aimed to develop an efficient, rapid, accurate, and sensitive detection method that is applicable for routine clinical use.MethodsEscherichia coli was used as a model organism to develop a rapid, accurate, and reliable multiplex polymerase chain reaction (M-PCR) for the detection of four aminoglycoside modifying enzyme (AME) resistance genes Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia. M-PCR was used to detect the distribution of AME resistance genes in 237 clinical strains of E. coli. The results were verified by simplex polymerase chain reaction (S-PCR).ResultsResults of M-PCR and S-PCR showed that the detection rates of Aac(6′)-Ib, Aac(3)-II, Ant(3″)-Ia, and Aph(3′)-Ia were 32.7%, 59.2%, 23.5%, and 16.8%, respectively, in 237 clinical strains of E. coli. Compared with the traditional methods for detection and identification, the rapid and accurate M-PCR detection method was established to detect AME drug resistance genes. This technique can be used for the clinical detection as well as the surveillance and monitoring of the spread of those specific antibiotic resistance genes.

Event-specific multiplex PCR method for four genetically modified cotton varieties, and its application

Multiplex polymerase chain reaction (PCR) methods have been developed and validated for screening, tracing, and regulating genetically modified (GM) crops in quarantine and environmental monitoring. In this study, we aimed to develop a method to simultaneously detect four GM cotton varieties in order to establish a screening system for cotton volunteers. Based on the sequence of DNA in the junction between introduced gene and flanking genomic DNA of four GM cotton events, herbicide-tolerant MON88701 and DAS-81910-7 and insect-resistant COT102 and T304-40, event-specific primers were designed and a multiplex detection method was developed. The simplex PCR results supported the multiplex PCR results; the amplification efficiency of the novel multiplex PCR method was increased compared with that of the Joint Research Centre (JRC) method. Based on the accuracy and efficiency, the method can be applied to detect and identify randomly mixed reference materials and suspected cotton volunteers. To apply this multiplex PCR method to living modified (LM) environmental monitoring samples, we performed additional PCR analysis to identify whether the volunteers were the four LM cotton varieties. As a result, 66 cotton volunteers were identified with stack event, comprising one or two of the four LM cotton events, and all stacks have been approved in South Korea for food, feed, and processing. These results indicated that our novel multiplex method is suitable for LMO identification.

Comparison of Two Multiplex PCR Systems for Meat Species Authentication

Background: Meat species adulteration has become a problem of concern. This study aimed to compare two previously published multiplex Polymerase Chain Reaction (PCR) methods for meat species authentication. 
 Methods: The primers used in the first multiplex PCR involved species-specific reverse primer for sheep, goat, cattle, pig, and donkey with universal forward primer. In the second multiplex PCR, the primers included species-specific forward and reverse primer for pork, lamb, ostrich, horse, and cow. The extracted DNA was then amplified with species-specific primers and with mix primers separately in the respective multiplex PCR.
 Results: The first multiplex PCR was accompanied with cross reactivity, whereas the second multiplex PCR was specific as expected for pork, lamb, ostrich, horse, and cow. The first set of multiplex PCR showed not always amplification of all species-specific DNAs with a mixture of DNA from mentioned animals. Regarding the second set of primers, the extracted DNA of different meat species was amplified with corresponding species primers as simplex PCR resulting in specific amplicons for species DNA prepared from sheep, ostrich, horse, pig, and cattle with the specific PCR products of 119, 155, 253, 100, and 311 bp, respectively.
 Conclusion: Based on the present investigation, we recommend the multiplex PCR with the second set of primers included species-specific forward and reverse primers for species authentication of five meat types, including pork, lamb, ostrich, horse, as well as cow.

Clinico- bacteriological study and molecular detection of campylobacter in childhood diarrhoea

Purpose: Campylobacter enteritis is rapidly becoming the most commonly recognized cause of bacterial gastroenteritis in man and causes 5–14% of diarrhea, worldwide. High incidence of Campylobacter diarrhoea, as well as its duration and possible sequelae, make it important from a socio-economic perspective. Diagnoses of Campylobacter infections are challenging because the organism is difficult to isolate, grow and identify. Guidelines for Clinical and laboratory diagnosis are inadequate. Methods & Materials: A Hospital based Cross sectional descriptive study conducted in JSS tertiary care hospital, Mysuru. Study included 55 stool samples of children aged between 1 to 60 months who presented to Department of Pediatrics, JSS hospital, both as in and out patients with complaints of diarrhea and dysentery. All stool samples were inoculated on to selective and non-selective media with filtration using a 0.45 μm membrane filters and incubated in microaerophilic conditions using the candle jar at temperatures 37 °C and 42 °C. The culture isolates were confirmed by standard phenotypic tests. A simplex polymerase chain reaction (PCR) with universal Campylobacter primers and primers specific for C.jejuni and C.coli, was performed on the DNA extracted from the stool filtrates. Results: Campylobacter was isolated in 5(9.1%) out of 55 stool samples using culture and the isolate were confirmed to be Campylobacter jejuni by phenotypic tests. Campylobacter Genus level PCR was positive for 10 samples (18.2%), rest of the samples were negative. The positive samples were subjected to species level PCR and all were positive for C.jejuni (100%) and negative for C.coli (0%). Conclusion: The implication of this study is that culture is less sensitive for use in diagnosis of Campylobacter infection in our settings. Nucleic acid based diagnostics offer increased sensitivity, can determine both the presence and burden of infection, and can distinguish between Campylobacter infections at the species level. We therefore recommend PCR, if feasible, as the preferred diagnostic modality for detection of Campylobacter infection. However, the role of culture in the diagnosis of Campylobacter infections is that it allows precise identification of bacteria and testing of their susceptibilities, which are integral to management of patients.

A duplex PCR for the simultaneous detection of Fasciola hepatica and Clonorchis sinensis

Both Fasciola hepatica and Clonorchis sinensis are endemic in China, South Korea, Japan and other Southeast Asian countries. Reliable and sensitive diagnostic methods are needed for detecting their infections in humans and animals. Differential simplex and duplex polymerase chain reaction (PCR) methods were developed. The PCRs targeted the second internal transcribed spacer (its2) (408 bp) of F. hepatica, and NADH dehydrogenase subunit 2 gene (nad2) (527 bp) of C. sinensis. Both simplex PCRs detected as little as 2 pg genomic DNA in one microliter in a 25 μL PCR reaction system. The duplex PCR had similar detection limit as well, and detected as low as one egg in 200 mg feces. These methods were analytical specific with no amplification being observed from the gemonic DNA of Fasciolopsis buski, Haemonchus contortus, Ascaris ovis or Eimeri ahsata. Of 158 sheep fecal samples collected from various farms, four and one samples were PCR-positive for F. hepatica and C. sinensis, respectively. The duplex PCR method described here is time-saving and convenient, and may prove to be an invaluable tool for molecular detection and epidemiological investigation of F. hepatica and C. sinensis in endemic area.

Antimicrobial resistance in Escherichia coli and Salmonella spp. isolates from fresh produce and the impact to food safety

ABSTRACTFoodborne diseases associated with fresh produce consumption have escalated worldwide, causing microbial safety of produce of critical importance. Bacteria that have increasingly been detected in fresh produce are Escherichia coli and Salmonella spp., both of which have been shown to progressively display antimicrobial resistance. The study focused on the assessment of antimicrobial resistance of these enteric bacteria from different kinds of fresh produce from various open air markets and supermarkets in the Philippines. Using the disk diffusion assay on a total of 50 bacterial isolates obtained from 410 fresh produce surveyed, monoresistance to tetracycline was observed to be the most prevalent (38%), followed by multidrug resistance to tetracycline, chloramphenicol, ciprofloxacin, and nalidixic acid (4%), and lastly by dual resistance to tetracycline and chloramphenicol (2%). Using multiplex and simplex polymerase chain reaction (PCR) assays, tetA (75%) and tetB (9%) were found in tetracycline resistant isolates, whereas catI (67%) and catIII (33%) were detected in chloramphenicol resistant isolates. Sequence analysis of gyr and par genes from the ciprofloxacin and nalidixic acid resistant isolates revealed different mutations. Based on the results, fresh produce act as a reservoir of these antibiotic resistant bacteria which may pose health threat to consumers.

Simultaneous detection of bovine and porcine DNA in pharmaceutical gelatin capsules by duplex PCR assay for Halal authentication

BackgroundIn the pharmaceutical industry, hard- and soft-shelled capsules are typically made from gelatin, commonly derived from bovine and porcine sources. To ensure that pharmaceutical products comply with halal regulations in Muslim countries (no porcine products allowed), development of a valid, reliable, quick, and most importantly, cost-effective tests are of utmost importance.MethodsWe developed a species-specific duplex polymerase chain reaction (PCR) assay targeting 149 bp porcine and 271 bp bovine mitochondrial DNA (mtDNA) to simultaneously detect both porcine and bovine DNA (in one reaction at the same time) in gelatin. Some additional simplex PCR tests (targeting 126 bp bovine and 212 bp porcine mtDNA) and real-time PCR using a commercially available kit (for identification of porcine DNA) were used to verify the selectivity and sensitivity of our duplex PCR. After optimization of DNA extraction and PCR methods, hard/soft pharmaceutical gelatin capsules (containing drug) were tested for the presence of porcine and/or bovine DNA.ResultsDuplex PCR detected the presence of as little as 0.1% porcine DNA, which was more accurate than the commercially available kit. Of all gelatin capsules tested (n = 24), 50% contained porcine DNA (pure porcine gelatin alone or in combination with bovine gelatin).ConclusionsDuplex PCR presents an easy-to-follow, quick, low-cost and reliable method to simultaneously detect porcine and bovine DNAs (>100 bp) in minute amounts in highly processed gelatin-containing pharmaceutical products (with a 0.1% sensitivity for porcine DNA) which may be used for halal authentication.Graphical abstractSimultaneous detection of porcine and bovine DNA in gelatin capsules by duplex PCR

Occurrence of Vibrio Parahaemolyticus, Vibrio Cholerae and Vibrio Vulnificus in the Clam Ruditapes Philippinarum (Adams & Reeve, 1850) from Emilia Romagna and Sardinia, Italy.

Marine vibrios, Vibrio parahaemolyticus, V. vulnificus and V. cholerae are responsible of the majority of food-borne human infections by consumption of bivalve shellfish. The aim of the present study was to ascertain the occurrence of these bacteria, and their potential pathogenicity, in the Manila clam R. philippinarum from Emilia Romagna (ER) and Sardinia (SR) regions, Italy. Isolation was performed on CHROMagarTM vibrio with subculture on (thiosulfate-citrate-bile salts-sucrose) Agar and m-modified-cellobiose-polymyxin b-colistin (-CPC) Agar. Suspected strains were purified, biochemically characterized and genotyped by simplex polymerase chain reaction (PCR) for the specie-specific and pathogenic gene markers: V. parahaemolyticus (toxRP, tdh and trh); V. vulnificus (vvhA, hsp, vcgC, vcgE, CPS operon allele 1, CPS operon allele 2, 16s-rRNA operon allele A, 16s-rRNA operon allele B; V. cholerae (toxRC, hlya, tcpI, tcpA, ctxA, ctxB, stn/sto). Moreover a multiplex PCR was applied to the SR bivalve shellfish, for the simultaneous detection of the three targets directly on homogenate samples, targeting the species-specific gene for V. cholerae (toxRC), V. parahaemolyticus (toxRP) and V. vulnificus (vvhA). As a result of phenotyping and genotyping of isolates, bivalve shellfish from ER resulted positive for V. parahaemolyticus (27.8%) and V. vulnificus (10.1%), but negative for V. cholerae. Shellfish from SR resulted positive for V. parahaemolyticus (30.3%), V. vulnificus (6.1%) and V. cholerae (3%). No significant differences emerged between the two areas (P>0.05).

A Rapid and Cost-Efficient Technique for Simultaneous/Duplex Detection of Listeria Monocytogenes and Escherichia Coli O157:H7 Using Real Time PCR

The increasing industrial interest in easy, economical and reliable methods has led to the development and application of DNA-based methods for the detection of microbial pathogens in food. In the present article, we describe the development of a cost-efficient EvaGreen-based real-time Polymerase chain reaction (PCR) technique for simultaneous and practical detection of Listeria monocytogenes (L. monocytogenes) and Escherichia coli O157:H7 (E. coli O157:H7) in the food matrix. EvaGreen-based simplex and duplex real-time PCR showed that specific PCR products were identified by melting curve analysis, and had a reproducible Tm of 77.00 ± 0.4C for L. monocytogenes and 85.60 ± 0.2 for E. coli O157:H7.The absolute detection limit of Eva Green-based simplex and duplex real-time PCR was 0.0001 ng/μL for DNA of both pathogens. The relative detection limit of the simplex and duplex technique was less than 10 cells/mL for the two pathogens. Practical Applications This study illustrates a rapid, effective and cheap identification method to simultaneously monitor two foodborne pathogens in food samples. The specificity and sensitivity of this method can be used to clearly identify these two foodborne pathogens in practical food samples based on the specific genes of the species. Hence, this detection method is applicable to surveillance measures for these two foodborne pathogens in the food production chain. Moreover, this technique is used by food control laboratories in a secure way.

A lab-on-a-chip-based multiplex platform to detect potential fraud of introducing pig, dog, cat, rat and monkey meat into the food chain

Food forgery has posed considerable risk to public health, religious rituals, personal budget and wildlife. Pig, dog, cat, rat and monkey meat are restricted in most religions, but their sporadic adulteration are rampant. Market controllers need a low-cost but reliable technique to track and trace suspected species in the food chain. Considering the need, here we documented a lab-on-a-chip-based multiplex polymerase chain reaction (PCR) assay for the authentication of five non-halal meat species in foods. Using species-specific primers, 172, 163, 141, 129 and 108-bp sites of mitochondrial ND5, ATPase 6 and cytochrome b genes were amplified to detect cat, dog, pig, monkey and rat species under complex matrices. Species-specificity was authenticated against 20 different species with the potential to be used in food. The targets were stable under extreme sterilisation (121°C at 45 psi for 2.5 h) which severely degrades DNA. The assay was optimised under the backgrounds of various commercial meat products and validated for the analysis of meatballs, burgers and frankfurters, which are popular fast food items across the globe. The assay was tested to detect 0.1% suspected meats under commercial backgrounds of marketed foods. Instead of simplex PCR which detects only one species at a time, such a multiplex platform can reduce cost by at least fivefolds by detecting five different species in a single assay platform.

Phenotypic and genotypic detection of virulence factors of Staphylococcus aureus isolated from clinical and subclinical mastitis in cattle and water buffaloes from different farms of Sadat City in Egypt.

Aim:To characterize Staphylococcus aureus from clinical and subclinical mastitis and identify virulence factors.Materials and Methods:Two hundred and two milk samples were collected, 143 from mastitic cattle and buffaloes 94 and 49, respectively, and 59 from apparently healthy cattle and buffaloes 35 and 24, respectively.Results:California mastitis test was applied and positive prevalence were 91.48% and 75.51% for cattle and buffalo with clinical mastitis and 37.14% and 45.83% for cattle and buffalo with subclinical mastitis. S. aureus was isolated from clinically mastitic cattle and buffaloes were 39.29% and 50%, respectively. While, from subclinical mastitic cattle and buffaloes were 80% and 72.73%, respectively. Hemolytic activity was assessed for S. aureus isolated from clinically and subclinical mastitic cases with prevalences of 100% and 56.25%, respectively. Thermo nuclease production from clinically and subclinical mastitic cases was 25% and 56.25%, respectively. Simplex polymerase chain reaction (PCR) conducted on S. aureus using 16S rRNA, clumping factor A, Panton valentine leukocidin, coagulase (Coa), alpha-hemolysin and beta-hemolysin those proved existence in 100%, 46.9%, 65.6%, 100%, 34.4%, and 43.75% of the isolates, respectively. While, multiplex PCR is utilized for detection of enterotoxins and proved that 12.5% was positive for enterotoxine Type D.Conclusions:It is concluded that simplex and multiplex PCR assays can be used as rapid and sensitive diagnostic tools to detect the presence of S. aureus and characterize its virulence factors that help in detection of severity of infection, distribution and stating preventive and control strategies.