Potential For Field Use Research Articles

Rapid and sensitive detection of Enterocytozoon hepatopenaei in real-time recombinase polymerase amplification assay

Enterocytozoon hepatopenaei (EHP) is a very serious pathogen in cultured shrimp, which brings huge economic losses to the shrimp industry every year. In this study, we established a real-time recombinase polymerase amplification (real-time RPA) assay based on the spore wall protein (SWP) gene sequence for the quantitative detection of the shrimp microsporidian EHP. The real-time RPA assay results indicated a high sensitivity and no cross-reaction with other aquatic pathogens. The limit of detection (LOD) was as low as 174 copies/μL in 95% of cases, as determined by probit analysis of eight independent experiments. The established standard curves showed correlation coefficient values (R2 = 0.9418) in the range of 1.0 × 100 to 1.0 × 108 copies/uL. Fifty samples of Litopenaeus vannamei were collected in shrimp farms and subjected to detection with real-time RPA and qPCR. Eight negative and 42 positive samples were detected by real-time RPA. Compared with those of qPCR (43/50), the diagnostic sensitivity and specificity of real-time RPA were 97.7% and 100%, respectively. Consequently, this rapid real-time RPA method has great application potential for field use or point-of-care diagnostics.

Rapid detection of CITES-listed shark fin species by loop-mediated isothermal amplification assay with potential for field use

Shark fin is a delicacy in many Asian countries. Overexploitation of sharks for shark fin trading has led to a drastic reduction in shark population. To monitor international trade of shark fin products and protect the endangered species from further population decline, we present rapid, user-friendly and sensitive diagnostic loop-mediated isothermal amplification (LAMP) and effective polymerase chain reaction (PCR) assays for all twelve CITES-listed shark species. Species-specific LAMP and PCR primers were designed based on cytochrome oxidase I (COI) and NADH2 regions. Our LAMP and PCR assays have been tested on 291 samples from 93 shark and related species. Target shark species could be differentiated from non-target species within three hours from DNA extraction to LAMP assay. The LAMP assay reported here is a simple and robust solution for on-site detection of CITES-listed shark species with shark fin products.

Recent advances in SERS detection of perchlorate

Perchlorate has recently emerged as a widespread environmental contaminant of healthy concern. Development of novel detection methods for perchlorate with the potential for field use has been an urgent need. The investigation has shown that surface-enhanced Raman scattering (SERS) technique has great potential to become a practical analysis tool for the rapid screening and routine monitoring of perchlorate in the field, particularly when coupled with portable/handheld Raman spectrometers. In this review article, we summarize progress made in SERS analysis of perchlorate in water and other media with an emphasis on the development of SERS substrates for perchlorate detection. The potential of this technique for fast screening and field testing of perchlorate-contaminated environmental samples is discussed. The challenges and possible solutions are also addressed, aiming to provide a better understanding on the development directions in the research field.

Development of Screen Printed Electrodes Chemically Modified with Schiff Base Films for Application in Forensic Chemistry

Electrochemical techniques have often been used to analyze a broad range of substances of forensic importance. These techniques can offer certain operational advantages such as ease of automation, potential for field use and the capability of in vivo analysis. They can also furnish information about reaction mechanisms. Additionally, chemical modification of conventional electrode surface is commonly employed for increasing specificity or selectivity in drug analysis. In the case of cocaine, for example, a variety of modifiers have been used, including polymers, Schiff bases, and biological materials. In this work, chemically modified screen printed electrodes (CM-SPE) using different Schiff base films were employed in the voltammetric investigation of cocaine, 3,4 - methylenedioxymethanphetamine (MDMA), and morphine. Dip coating process was chosen for the surface chemical modification. Three different electrode plateforms were employed: carbon, gold, and platinum. with methanolic solution containing different Schiff base complexes with subsequent drying step at room temperature. The voltammetric measurements were carried out in 50:50 v/v methanol:water solution containing 0.05 mol L-1 KCl and 0.1 mol L-1 LiCl as a supporting electrolyte without oxygen elimination by inert gas flow. The best results were obtained with carbon electrodes. Cocaine exhibits a well defined irreversible anodic peak current (ipa) at a potential (Epa) of 0.85V vs Ag/AgCl. Voltammetric parameters such as Eprec, v, and tprec were optimized in this study, being obtained the values of -0.80V (vs Ag/AgCl), 120 s, and 100 mV s-1, respectively. The optimized amount of the chemical modifier was found to be 30 mg per electrode. Detection and quantification limits were obtained in 110 and 390 mmol L-1, respectively. Additionally, intra and inter-day repeatability of 2.61% and 3.77%, were obtained for cocaine determination. It was possible to obtain a transductor for cocaine in presence of morphine and MDMA with high specificity in trace levels. The author acknowledges FAPESP (2013/17630-7) and CAPES (25/2014 Pro-Forenses) for financial suport.

Rapid detection of shrimp white spot syndrome virus by real time, isothermal recombinase polymerase amplification assay.

White spot syndrome virus (WSSV) causes large economic losses to the shrimp aquaculture industry, and thus far there are no efficient therapeutic treatments available against this lethal virus. In this study, we present the development of a novel real time isothermal recombinase polymerase amplification (RPA) assay for WSSV detection on a small ESEQuant Tube Scanner device. The RPA sensitivity, specificity and rapidity were evaluated by using a plasmid standard as well as viral and shrimp genomic DNAs. Compared with qPCR, the RPA assay revealed more satisfactory performance. It reached a detection limit up to 10 molecules in 95% of cases as determined by probit analysis of 8 independent experiments within 6.41±0.17 min at 39°C. Consequently, this rapid RPA method has great application potential for field use or point of care diagnostics.

Recombinase Polymerase Amplification Assay for Rapid Detection of Francisella tularensis

Several real-time PCR approaches to develop field detection for Francisella tularensis, the infectious agent causing tularemia, have been explored. We report the development of a novel qualitative real-time isothermal recombinase polymerase amplification (RPA) assay for use on a small ESEQuant Tube Scanner device. The analytical sensitivity and specificity were tested using a plasmid standard and DNA extracts from infected rabbit tissues. The assay showed a performance comparable to real-time PCR but reduced the assay time to 10 min. The rapid RPA method has great application potential for field use or point-of-care diagnostics.

Peripheral Blood Buffy Coat Smear: a Promising Tool for Diagnosis of Visceral Leishmaniasis

Confirmative diagnosis of visceral leishmaniasis (VL) is still a challenge at the primary health care facilities in most of the rural areas of endemicity in the Indian subcontinent. Conventional methods for parasitological confirmation are risky and require skilled personnel, and hence they are unavailable to the poor people in the regions of endemicity. Buffy coat smear microscopy, as a minimally invasive, simple alternative for the parasitological diagnosis of VL, was evaluated in this prospective study. One hundred twelve VL patients were enrolled in this study. The buffy coat was separated from peripheral blood of all enrolled subjects using Histopaque-1119 solution. Leishman-stained buffy coat smears were examined for Leishmania donovani bodies, and buffy coat was also utilized for detection of parasite DNA by Leishmania nested PCR (LnPCR) for all cases. Concomitant splenic smears could be examined for L. donovani bodies in 66 cases, and the parasite load was graded on a scale of 1+ to 6+ for L. donovani-positive smears. All splenic smear-positive cases were also found to be positive by LnPCR. Of 112 enrolled VL cases, 103 (92%) were found to be positive for L. donovani bodies in buffy coat smear microscopy, which is promising as a confirmative diagnosis tool. We have also found a significant association of the buffy coat smear positivity with parasitic burden in the spleen smear. In this preliminary observation in Bangladesh, buffy coat smear microscopy has been found to be very simple, minimally invasive, and risk-free method of parasitological diagnosis of VL with a good diagnostic accuracy and potential for field use.

Baseline Sensitivity of Fusarium Species Associated with Fusarium Diseases to Metconazole, Triticonazole, and Thiabendazole Fungicides

Baseline Sensitivity of Fusarium Species Associated with Fusarium Diseases to Metconazole, Triticonazole, and Thiabendazole Fungicides

Dengue fever in the Western Hemisphere.

Dengue virus, an arthropod-borne viral agent, causes two distinct diseases: classic dengue fever (DF) and dengue hemorrhagic fever (DHF). There are four dengue virus serotypes: DEN-1, DEN-2, DEN-3, and DEN-4. Although infection with dengue stimulates immunologic response to a serotype, there is no cross-immunity conferred. Hence, a person can potentially be infected with each serotype during his or her lifetime. An infected female Aedes mosquito transmits the virus from person to person while feeding. The disease, now endemic in more than 100 countries in Africa, the Americas, the Eastern Mediterranean, Southeast Asia, and Western Pacific, is spreading to new areas and causing explosive outbreaks. Because of the major impact on lives and local economies epidemics produce, rapid detection of dengue infection has become an important public health research issue. Recently developed serological procedures to detect dengue infections have shown great potential for field use.

Field application of PCR-based assays for monitoring Wuchereria bancrofti infection in Africa.

Approximately 50 million people in Egypt and sub-Saharan Africa have bancroftian filariasis and together they represent about a third of all cases of lymphatic filariasis (LF) world-wide. Currently, the Global Programme to Eliminate Lymphatic Filariasis, which was launched by the World Health Organization (WHO) in 1998, is largely based on repeated annual cycles of mass drug administration (MDA) to endemic populations. Also, some countries, including Egypt, are taking steps to improve LF vector-control interventions, to break the transmission cycle more effectively than is achievable with MDA alone. New tools and strategies for monitoring and evaluating elimination campaigns are needed. The last 20 years have witnessed dramatic advances in the diagnosis of LF for epidemiological purposes. The recent introduction and development of molecular technologies have moved parasite-detection systems from traditional methods (that are labour-intensive, tedious and often impractical) to improved PCR-based assays that have considerable potential for field use. The present article highlights the strengths and limitations of the PCR-based assays when used to detect filarial infections in mosquitoes (particularly for the xenomonitoring of elimination campaigns).

Recent advances in molecular diagnostic techniques for human lymphatic filariasis and their use in epidemiological research

Diagnosis of lymphatic filariasis for epidemiological studies is an area of science that has been greatly modernized over the last two decades. Recent developments and the introduction of new technologies have led to the replacement of traditional diagnostic methods that were insensitive, tedious and often impractical with molecular biology techniques that have considerable potential for field use. This paper reviews some of these major new techniques and the applications of molecular diagnosis in the field of lymphatic filariasis.

Ribosomal RNA: Nature's own polymerase-amplified target for diagnosis

The technological criteria for diagnostic tests for tropical diseases are strict and limited. One method of considerable potential for field use is the exploitation of regions of species-specific sequences of ribosomal RNA (rRNA). Oligodeoxynucleotide probes can be used to detect the specific RNa regions in question. Andy Waters and Tom McCutchan describe how simple rRNA methodologies for Plasmodium can detect and identify parasites as well as follow different stages in their development. The potential of this methodology extends to monitoring both epidemiological parameters and the efficacy of novel drug regimes.

The development of nonradioactive total genomic probes for strain and egg differentiation in taeniid cestodes.

Purified total genomic DNA labeled with photobiotin was used as a probe for the identification and differentiation of strains of Echinococcus granulosus and to distinguish between the morphologically identical eggs of 2 taeniid species. Such biotinylated probes are easily prepared and can be utilized effectively for rapid screening and identification of taeniid isolates by dot and Southern blotting. The use of total genomic DNA probes in high stringency hybridizations obviates the need for cloned sequences and has great potential for field use.