Development of HRPzyme-Integrated PCR Platform for Colorimetric Detection of Foodborne Pathogens

In recent years, foodborne illnesses have become the most significant public health issue in both developed and developing countries. The World Health Organization (WHO) reported that in 2010, around 1.8 million people died due to foodborne illness. Therefore, the development of a cost-effective, sensitive, and selective detection method for identifying and monitoring foodborne pathogens is necessary for improved public health. Here, we describe a simple and ultrasensitive colorimetric method for the detection of foodborne pathogens based on HRPzyme-integrated PCR using PC-based ImageJ software. We present insights into different aspects of this method such as the importance of 16S rRNA detection, the modification of traditional PCR primers with a unique functional sequence for generating a color signal, and the application of ImageJ in colorimetric image data acquisition. The performance of the proposed strategy in detecting various foodborne pathogens is comparable to that of the commercial UV-Vis spectrophotometer Tecan Infinite 200 Pro. This detection platform exhibits linearity over wide range, high sensitivity, and high selectivity. The diagnostic capability of this colorimetric system to detect foodborne pathogens was demonstrated with spiked fruit and vegetable samples. This low-cost and effective colorimetric method can be conveniently employed for the analysis of DNA sequences arising from pathogenic bacteria

Bioconjugated nanomaterials for monitoring food contamination

Maintaining food safety and hygiene standards is top priority and challenge for farmers, food industries, governments and food technologists working in the food supply chain. Pesticides, toxins, veterinary drug residues, foodborne pathogens and many other harmful chemicals that may be present in a vast array of food products, due to various stages of their production like packaging and transport, constitute a global health problem that requires powerful and innovative technologies allowing constant and accurate detection of food products from production to consumption. Recent progress in generation of specific synthetic oligonucleotides against food contaminants has provided a new insight into the current sensor technologies, where these functional synthetic oligonucleotides, so-called aptamers, have been successfully combined with nanomaterials for rapid and cost-effective detection of several substances related to the food contamination, such as antibiotics, mycotoxins, heavy metals, carcinogenic dyes, pesticides, pathogens and other plastic products used for food packaging. Unique characteristics of aptamers over antibodies, such as in vitro selection, chemical and thermal stability, small size and ease of labeling have laid the solid foundation for exploring aptamers further in multiplexed food monitoring systems. In this chapter, we reviewed the application of aptamer-conjugated nanomaterials in food safety surveillance as well as the conventional techniques used for food safety monitoring in order to provide a comprehensive and comparative approach

Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria

Foodborne pathogenic bacteria present a crucial food safety issue. Conventional diagnostic methods are time-consuming and can be only performed on previously produced food. The advancing field of point-of-need diagnostic devices integrating molecular methods, biosensors, microfluidics, and nanomaterials offers new avenues for swift, low-cost detection of pathogens with high sensitivity and specificity. These analyses and screening of food items can be performed during all phases of production. This review presents major developments achieved in recent years in point-of-need diagnostics in land-based sector and sheds light on current challenges in achieving wider acceptance of portable devices in the food industry. Particular emphasis is placed on methods for testing nucleic acids, protocols for portable nucleic acid extraction and amplification, as well as on the means for low-cost detection and read-out signal amplification

Paper-based microfluidics for rapid diagnostics and drug delivery

Paper is a common material that is promising for constructing microfluidic chips (lab-on-a-paper) for diagnostics and drug delivery for biomedical applications. In the past decade, extensive research on paper-based microfluidics has accumulated a large number of scientific publications in the fields of biomedical diagnosis, food safety, environmental health, drug screening and delivery. This review focuses on the recent progress on paper-based microfluidic technology with an emphasis on the design, optimization and application of the technology platform, in particular for medical diagnostics and drug delivery. Novel advances have concentrated on engineering paper devices for point-of-care (POC) diagnostics, which could be integrated with nucleic acid-based tests and isothermal amplification experiments, enabling rapid sample-to-answer assays for field testing. Among the isothermal amplification experiments, loop-mediated isothermal amplification (LAMP), an extremely sensitive nucleic acid test, specifically identifies ultralow concentrations of DNA/RNA from practical samples for diagnosing diseases. We thus mainly focus on the paper device-based LAMP assay for the rapid infectious disease diagnosis, foodborne pathogen analysis, veterinary diagnosis, plant diagnosis, and environmental public health evaluation. We also outlined progress on paper microfluidic devices for drug delivery. The paper concludes with a discussion on the challenges of this technology and our insights into how to advance science and technology towards the development of fully functional paper devices in diagnostics and drug delivery

Biosensors Based on Isothermal DNA Amplification for Bacterial Detection in Food Safety and Environmental Monitoring

The easy and rapid spread of bacterial contamination and the risk it poses to human health makes evident the need for analytical methods alternative to conventional time-consuming laboratory-based techniques for bacterial detection. To tackle this demand, biosensors based on isothermal DNA amplification methods have emerged, which avoid the need for thermal cycling, thus facilitating their integration into small and low-cost devices for in situ monitoring. This review focuses on the breakthroughs made on biosensors based on isothermal amplification methods for the detection of bacteria in the field of food safety and environmental monitoring. Optical and electrochemical biosensors based on loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), recombinase polymerase amplification (RPA), helicase dependent amplification (HDA), strand displacement amplification (SDA), and isothermal strand displacement polymerisation (ISDPR) are described, and an overview of their current advantages and limitations is provided. Although further efforts are required to harness the potential of these emerging analytical techniques, the coalescence of the different isothermal amplification techniques with the wide variety of biosensing detection strategies provides multiple possibilities for the efficient detection of bacteria far beyond the laboratory bench.info:eu-repo/semantics/publishedVersio

Host-selected mutations converging on a global regulator drive an adaptive leap towards symbiosis in bacteria

Host immune and physical barriers protect against pathogens but also impede the establishment of essential symbiotic partnerships. To reveal mechanisms by which beneficial organisms adapt to circumvent host defenses, we experimentally evolved ecologically distinct bioluminescent Vibrio fischeri by colonization and growth within the light organs of the squid Euprymna scolopes. Serial squid passaging of bacteria produced eight distinct mutations in the binK sensor kinase gene, which conferred an exceptional selective advantage that could be demonstrated through both empirical and theoretical analysis. Squid-adaptive binK alleles promoted colonization and immune evasion that were mediated by cell-associated matrices including symbiotic polysaccharide (Syp) and cellulose. binK variation also altered quorum sensing, raising the threshold for luminescence induction. Preexisting coordinated regulation of symbiosis traits by BinK presented an efficient solution where altered BinK function was the key to unlock multiple colonization barriers. These results identify a genetic basis for microbial adaptability and underscore the importance of hosts as selective agents that shape emergent symbiont populations

CHARACTERISATION AND MOLECULAR TYPING OF CLINICAL AND ENVIRONMENTAL ISOLATES OF VIBRIO PARAHAEMOLYTICUS

Vibrio parahaemolyticus is a natural inhabitant of coastal waters worldwide and is the leading cause of seafood-borne gastroenteritis. This study reports on the use of several molecular characterisation methods to screen clinical and environmental isolates of V parahaemolyticus to assess whether such techniques can be used to distinguish pathogenic isolates reliably. In a total of 86 isolates mainly of V parahaemolyticus but also including V cholerae, V vulnificus and several other species, serotypes of the more virulent clonal group 03:K6 were identified, but otherwise there appeared no association with serotype and phenotype. The tdh and trh genes encoding haemolysins that are typically associated with virulent isolates were found in a significantly large number of isolates; however, poor concordance between haemolytic activity and the presence of the gene tdh was found. In an effort to establish more accurate relationships amongst clinical and environmental isolates of V parahaemolyticus, four molecular typing systems were employed; namely pulsed-field gel electrophoresis (PFGE), intergenic transcribed spacer (ITS) analysis, tDNA intergenic length polymorphisms (tDNA-ILPs) and randomly amplified polymorphic DNA (RAPD). Typing patterns and clustering analysis using these methods differentiated V parahaemolyticus from other marine species as well as at the subspecies level. PFGE with NotI was shown to be the most discriminative but suffered from not being universally applicable. Both ITS and tDNA-ILP methods were sufficiently discriminatory with discrimination indices (DI) of between 0.568 and 0.724, depending on the primers employed. The discriminatory ability of RAPD was also affected by the primers used (DI= 0.959 - 0.965) but closely matched that of PFGE (DI = 0.976). Additionally, both RAPD methods were able to distinguish putative markers for the pandemic clonal group. Typing systems appeared largely stable in duplicate and triplicate analyses with multiple primer pairs with some obvious variability in the reproduction of faint amplicons. All methods except PFGE were simple to execute but none of the methods could distinguish V parahaemolyticus into obvious lineages based on the clinical or environmental source. With the recent implication of a type Ill secretion system {TTSS) involved in the pathogenicity of V parahaemolyticus, a multiplex PCR system using PCR primers that spanned both TTSSl and TTSS2 regions was developed. Dot-blot analysis confirmed TTSS2 genes in at least 30% of environmental isolates. Nucleotide sequence analysis revealed l00% sequence homology in three loci of TTSS2 putative structural genes. In comparison, a total of 34 single nucleotide polymorphisms (SNP) were identified in three TTSS1 regions. In two of the regions, the SNPs were synonymous, whereas a non-synonymous substitution in the structural gene vcrDI resulted in valine replacement with isoleucine. In addition, nucleotide deletions in TTSS1 with resultant frameshift mutations were identified. The finding that significant numbers of environmental isolates also possess TTSS2 genes is contrary to currently held opinion that TTSS2 is only present in clinical isolates. It is hypothesed that the high incidences of V parahaemolyticus infections may be related to active TTSS2 genes, whereas a high degree of polymorphisms in TTSS1 suggest it may be inactive.The Centre for Environment, Fisheries and Aquaculture Science Weymouth Laboratories, Dorset, United Kingdo

Molecular Techniques for the Detection of Organisms in Aquatic Environments, with Emphasis on Harmful Algal Bloom Species.

Molecular techniques to detect organisms in aquatic ecosystems are being gradually considered as an attractive alternative to standard laboratory methods. They offer faster and more accurate means of detecting and monitoring species, with respect to their traditional homologues based on culture and microscopic counting. Molecular techniques are particularly attractive when multiple species need to be detected and/or are in very low abundance. This paper reviews molecular techniques based on whole cells, such as microscope-based enumeration and Fluorescence In-Situ Hybridization (FISH) and molecular cell-free formats, such as sandwich hybridization assay (SHA), biosensors, microarrays, quantitative polymerase chain reaction (qPCR) and real time PCR (RT-PCR). Those that combine one or several laboratory functions into a single integrated system (lab-on-a-chip) and techniques that generate a much higher throughput data, such as next-generation systems (NGS), were also reviewed. We also included some other approaches that enhance the performance of molecular techniques. For instance, nano-bioengineered probes and platforms, pre-concentration and magnetic separation systems, and solid-phase hybridization offer highly pre-concentration capabilities. Isothermal amplification and hybridization chain reaction (HCR) improve hybridization and amplification techniques. Finally, we presented a study case of field remote sensing of harmful algal blooms (HABs), the only example of real time monitoring, and close the discussion with future directions and concluding remarks

Addressing acute hepatopancreatic necrosis disease (AHPND) and other transboundary diseases for improved aquatic animal health in Southeast Asia: Proceedings of the ASEAN Regional Technical Consultation on EMS/AHPND and Other Transboundary Diseases for Improved Aquatic Animal Health in Southeast Asia, 22-24 February 2016, Makati City, Philippines

The editors would like to thank the members of SEAFDEC/AQD’s Publications Review Committee for reviewing the Proceedings prior to its publication. Thanks are also due to the Development Communication Section of the Training and Information Division of SEAFDEC/AQD, led by Mr. Rex Dianala, for copy-editing and layout. And lastly, to Mr. Isidro Tendencia for the design and concept of the cover.Collapse All ExpandAllFOREWORDMESSAGESACKNOWLEDGEMENTSPHOTO OF PARTICIPANTSBACKGROUNDPOLICY RECOMMENDATIONSREVIEW PAPERSLatest Research on Acute Hepatopancreatic Necrosis Disease (AHPND) of Penaeid ShrimpsIkuo Hirono, Sasiwipa Tinwongger, Yuki Nochiri, and Hidehiro KondoOIE Initiatives on Acute Hepatopancreatic Necrosis Disease (AHPND) and Other Aquatic Animal Diseases in AsiaHirofumi KugitaAcute Hepatopancreatic Necrosis Disease (AHPND) of Penaeid Shrimps: Global PerspectiveMelba G. Bondad-ReantasoRegional Response on AHPND and Other Emerging Shrimp Diseases in the Asia-PacificEduardo M. LeañoCOUNTRY PAPERSCambodiaCurrent Status of Shrimp Farming and Diseases in CambodiaOuch Lang and Mey SotheaIndonesiaCurrent Status of Acute Hepatopancreatic Necrosis Disease (AHPND) and Other Transboundary Diseases of Farmed Shrimps in IndonesiaMukti Sri Hastuti and DesrinaJapanImportant Diseases and Practical Control Measures in Shrimp Culture in JapanKei Yuasa, Toru Mekata, and Jun SatoLao PDRStatus of Aquatic Animal Health Activities in Lao PDRVonsamay Dalasaen and Bouakeo Vong AmnathMalaysiaCurrent Status of Acute Hepatopancreatic Necrosis Disease (AHPND) of Farmed Shrimp in MalaysiaKua Beng Chu, Ahmad IAR, Siti Zahrah A, Irene J, Norazila J, Nik Haiha NY, Fadzilah Y, Mohammed M, Siti Rokhaiya B, M Omar and Teoh TPMyanmarStatus of Shrimp Health Management in MyanmarSaw Lah Paw Wah and Maw Maw ThanPhilippinesStatus of Acute Hepatopancreatic Necrosis Disease (AHPND) of Cultured Shrimps in the PhilippinesMaria Abegail G. Apostol-AlbaladejoSingaporeStatus of Transboundary Diseases of Penaeid Shrimps in SingaporeWong Yelin and Jiang JunhuiThailandCurrent Status and Impact of Early Mortality Syndrome (EMS)/ Acute Hepatopancreatic Necrosis Disease (AHPND) and Hepatopancreatic Microsporidiosis (HPM) Outbreaks in Thailand’s Shrimp FarmingPutth Songsangjinda and Jaree PolchanaViet NamStatus of Acute Hepatopancreatic Necrosis Disease (AHPND) and Other Emerging Diseases of Penaeid Shrimps in Viet NamNguyen The Thien, Nguyen Thi Lan Huong, Vo Dinh Chuong, Nguyen Thi Viet Nga, Pham Hong Quang, Bui Thi Viet Hang, and Nguyen Van LongANNEXESList of ParticipantsSummary of Workshop Discussions</div

Vibrios in the Environment: An Investigation of Environmental Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio cholerae

Vibrio parahaemolyticus, Vibrio vulnificus, and Vibrio cholerae are Gram negative bacteria that naturally occur in marine and estuarine environment, both as free-floating cells or attached to chitinous surfaces. Although Vibrio spp. are readily isolated from the environment, not all strains are virulent. Therefore, the ability to detect the presence of vibrios is vital, but determination of pathogenicity is equally important. The research reported here was focused on prevalence and characteristics of environmental Vibrio species and how the environment provides a natural ecosystem for human pathogens and reservoir of virulence factors. Those objectives were achieved by carrying out intensive sampling over three years, during which water, sediment, and oyster samples were collected from the Chesapeake Bay, Maryland. Detection and molecular characterization of Vibrio parahaemolyticus and Vibrio vulnificus were done and the diversity of V. parahaemolyticus and V. vulnificus isolates from individual oyster samples was investigated. The large-scale populations of V. parahaemolyticus and V. vulnificus in the Chesapeake Bay and smaller scale populations of individual oysters were analyzed, thereby providing a snapshot of V. parahaemolyticus and V. vulnificus distribution in the environment. Because antibiotic resistance is an increasing public health concern, antibiograms of V. parahaemolyticus and V. vulnificus isolates from environmental sources were done to determine antibiotic resistance patterns in environmental isolates. Detection and enumeration of Vibrio species are a concern since Vibrio spp. can enter viable by nonculturable (VBNC) state. Thus, new and improved Vibrio detection methods are needed. This this study the Cholera O1 and O139 SMART II test were investigated for potential use in detecting V. cholerae in ballast water treatment systems