Detection Of Various Pathogens Research Articles

Multiplexed Instrument-Free Bar-Chart SpinChip Integrated with Nanoparticle-Mediated Magnetic Aptasensors for Visual Quantitative Detection of Multiple Pathogens.

A portable multiplexed bar-chart SpinChip (MB-SpinChip) integrated with nanoparticle-mediated magnetic aptasensors was developed for visual quantitative instrument-free detection of multiple pathogens. This versatile multiplexed SpinChip combines aptamer-specific recognition and nanoparticle-catalyzed pressure amplification to achieve a sample-to-answer output for sensitive point-of-care testing (POCT). This is the first report of pathogen detection using a volumetric bar-chart chip, and it is also the first bar-chart chip using a "spinning" mechanism to achieve multiplexed bar-chart detection. Additionally, the introduction of the spin unit not only enabled convenient sample introduction from one inlet to multiple separate channels in the multiplexed detection, but also elegantly solved the pressure cross-interference problem in the multiplexed volumetric bar-chart chip. This user-friendly MB-SpinChip allows visual quantitative detection of multiple pathogens simultaneously with high sensitivity but without utilizing any specialized instruments. Using this MB-SpinChip, three major foodborne pathogens including Salmonella enterica, Escherichia coli, and Listeria monocytogenes were specifically quantified in apple juice with limits of detection of about 10 CFU/mL. This MB-SpinChip with a bar-chart-based visual quantitative readout has great potential for the rapid simultaneous detection of various pathogens at the point of care and wide applications in food safety, environmental surveillance, and infectious disease diagnosis.

Nanozyme-based lateral flow assay for the sensitive detection of Escherichia coli O157:H7 in milk

Lateral flow assay (LFA) has been applied in many fields due to its relative ease of use and cost-effectiveness. However, it has low sensitivity and its applications are limited. Probe materials play a significant role in improving the detection efficiency and sensitivity of LFA. In this study, by using concave palladium-platinum (Pd-Pt) nanoparticles as a nanozyme probe, we developed a sensitive LFA based on the sandwich format for qualitative and quantitative detection of Escherichia coli O157:H7. The sensitivity of the LFA was improved by applying the 3,3',5,5'-tetramethylbenzidine (TMB) substrate onto the test line where the nanozyme was accumulated in the presence of analytes. The nanozyme showed high catalytic performance toward TMB and greatly enhanced the signal intensity of the test line. The sensitivity of the nanozyme-based LFA was 9.0 × 102 cfu/mL in milk, which was 111-fold higher than that of traditional colloidal gold-based LFA. The proposed method has remarkable potential in the detection of various pathogens in real samples.

Optical Immunosensors for the Efficient Detection of Target Biomolecules

Recently, immunosensors have attracted attention because they are widely applied for the detection of various pathogens. Among the commonly used immunosensors, the optical immunosensor features prominently as an effective tool for the quantification of the amount of antibodies, antigens, or haptens in complex samples with high sensitivity and specificity. However, very few studies provide comprehensive overviews of optical immunosensors. In this review, we present various methods and applications of optical immunosensors in pathogen detection. We introduced a concise definition of optical immunosensors and the principle of using them for detection. We subsequently discuss the main categories of optical immunosensors and their application to the detection of pathogens, as well as their advantages and limitations. Recent publications from 2006 to 2015 on variously designed optical immunosensors have also been updated. We conclude the review with a brief summary and discuss future directions of optical immunosensors.

Detection of Salmonella Enterica with Magnetoelastic Biosensors in Wash Water Containing Clorox and Chlorine Dioxide

Magnetoelastic (ME) biosensors with phage-displayed oligopeptide probes have been demonstrated to be highly successful in rapid detection of various pathogens, including Salmonella enterica, on the fruit and vegetable surfaces. However, in lieu of testing each produce individually, it is advantageous to detect pathogens in the produce wash water for high-throughput analysis. The sanitizing guidelines of the United States Food and Drug Administration (FDA) suggest adding Clorox and chlorine dioxide to the produce wash water in processing plants to disinfect harmful food-borne pathogens on fruit and vegetable surfaces. Therefore, to determine the efficacy of our ME biosensors in testing for food-borne pathogens in produce wash water, we assessed the stability of our biosensors in the presence of Clorox and chlorine dioxide. Specifically, Enzyme Linked Immunosorbent Assay (ELISA) was used to study the potential effect of Clorox and chlorine dioxide on the S. Typhimurium capturing ability of our phage probe. At concentrations recommended by the FDA for Clorox (100 ppm free chlorine content) and by the United States Environmental Protection Agency for chlorine dioxide (4 ppm), we observed no negative effect of these chemicals on the stability and S. Typhimurium capturing ability of our phage-displayed oligopeptide probe. These data demonstrate the potential efficacy of using our ME biosensors with phage-displayed oligopeptide probes in produce wash waters to determine the presence of food-borne pathogens.

Detection of Salmonella Enterica with Magnetoelastic Biosensors in Wash Water Containing Clorox and Chlorine Dioxide

Magnetoelastic (ME) biosensors with phage-displayed oligopeptide probes have been demonstrated to be highly successful in rapid detection of various pathogens, including Salmonella enterica, on the fruit and vegetable surfaces. However, in lieu of testing each produce individually, it is advantageous to detect pathogens in the produce wash water for high-throughput analysis. The sanitizing guidelines of the United States Food and Drug Administration (FDA) suggest adding Clorox and chlorine dioxide to the produce wash water in processing plants to disinfect harmful food-borne pathogens on fruit and vegetable surfaces. Therefore, to determine the efficacy of our ME biosensors in testing for food-borne pathogens in produce wash water, we assessed the stability of our biosensors in the presence of Clorox and chlorine dioxide. Specifically, Enzyme Linked Immunosorbent Assay (ELISA) was used to study the potential effect of Clorox and chlorine dioxide on the S. Typhimurium capturing ability of our phage probe. At concentrations recommended by the FDA for Clorox (100 ppm free chlorine content) and by the United States Environmental Protection Agency for chlorine dioxide (4 ppm), we observed no negative effect of these chemicals on the stability and S. Typhimurium capturing ability of our phage-displayed oligopeptide probe. These data demonstrate the potential efficacy of using our ME biosensors with phage-displayed oligopeptide probes in produce wash waters to determine the presence of food-borne pathogens.

Comparative Evaluation of Multiplex Real-Time PCR Assays for Six Pathogens of Sexually Transmitted Infections

Background: The multiplex real-time PCR assay is a sensitive test for simultaneous detection of various pathogens of sexually transmitted infections (STIs). We evaluated the performance of two multiplex real-time PCR assays for six STI pathogens. Methods: DNA samples after being used to conduct PCR ...

Direct detection of various pathogens by loop-mediated isothermal amplification assays on bacterial culture and bacterial colony.

In this work, loop-mediated isothermal amplification based detection assay using bacterial culture and bacterial colony for various common pathogens direct detection had been established, evaluated and further applied. A total of five species of common pathogens and nine detection targets (tlh, tdh and trh for V.Parahaemolyticus, rfbE, stx1 and stx2 for E.coli, oprI for P.aeruginosa, invA for Salmonella and hylA for L.monocytogenes) were performed on bacterial culture and bacterial colony LAMP. To evaluate and optimize this assay, a total of 116 standard strains were included. Then, for each detected targets, 20 random selected strains were applied. Results were determined through both visual observation of the changed color by naked eye and electrophoresis, which increased the accuracy of survey. The minimum adding quantity of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45min with 25μl reaction volume. The detection limit of bacterial culture LAMP and PCR assay were determined to be 102 and 104 or 105CFU/reaction, respectively. No false positive amplification was observed when subjecting the bacterial -LAMP assay to 116 reference strains. This was the first report of colony-LAMP and culture-LAMP assay, which had been demonstrated to be a fast, reliable, cost-effective and simple method on detection of various common pathogens.

Development of Quantitative Real-time Recombinase Polymerase Amplification (qRT-RPA) Method for Quantitative Detection against Pathogenic Virus in Honeybee

재조합효소-중합효소 증폭법(RPA)은 등온에서 매우 빠른 반응속도를 보이는 유전자 증폭법으로 근래 여러 병원체의 검출법에 폭넓게 적용되고 있다. 본 연구는 kSBV의 빠른 검색을 위하여 kSBV 특이 실시간 재조합효소-중합효소 증폭법을 새로이 개발하였으며, 특이 cDNA로부터 2분 46초만에 kSBV특이 유전자의 존재를 확인할 수 있었다. 또한, 이를 바탕으로 목적 유전자의 정량이 가능한, 보편적 응용이 가능한 정량 실시간 재조합효소-중합효소 증폭법(quantitative real-time RPA)을 제안하였으며, 이 정량법이 현장시료에서 유용함을 입증하였다. 본 실험법은 꿀벌 질병체의 정량 검출 뿐 아니라, 일반 병원체의 정량 검출에서도 응용되기를 기대한다.

A Proof of Concept: Detection of Avian Influenza H5 Gene by a Graphene-Enhanced Electrochemical Genosensor.

The highly pathogenic avian influenza (HPAI) virus subtype H5N1 has been found to be the most virulent and deadliest virus strain, with capability of interspecies transmission to human infection. Most human HPAI H5N1 cases were diagnosed late in their illnesses for medical care, resulting in severe complications that led to death. In this study, a novel graphene-enhanced electrochemical DNA biosensor had been fabricated for the detection of polymerase chain reaction (PCR) amplicon derived from the haemagglutinin (H5) gene of the HPAI. The graphene-enhanced DNA biosensor showed excellent linear correlation between PCR amplicon concentration and amperometric signal with a correlation coefficient, r2 of 0.9987. The amperometric response of the proposed biosensor was compared with conventional gel electrophoresis while the feasibility of the proposed sandwich sensing platform was verified via dot blot assay. The results obtained indicate that the electrochemical DNA biosensing assay is significantly more sensitive (P < 0.05) and time efficient. This work serves as a proof of concept in hopes for further development of the graphene enhanced electrochemical DNA biosensor into a portable, on-site screening platform for point-of-care detection of various pathogens.

Scientific Symposium “Small Solution for Big Water-Related Problems: Innovative Microarrays and Small Sensors to Cope with Water Quality and Food Security”

This issue presents the conclusive results of two European Commission funded Projects, namely Universal Microarrays for the Evaluation of Fresh-water Quality Based on Detection of Pathogens and their Toxins (MicroAQUA) and Rationally Designed Aquatic Receptors (RADAR). These projects focused their activities on the quality of drinking water as an extremely important factor for public health of humans and animals. The MicroAQUA Project aimed at developing a universal microarray chip for the detection of various pathogens (cyanobacteria, bacteria, viruses and parasitic protozoa) and their toxins in waters. In addition, the project included the detection of select species of diatoms, which represent reliable bio-indicators to assess overall water quality. Large numbers of compounds are released into the environment; some of these are toxins such as endocrine disrupting compounds (EDCs) and can affect the endocrine, immune and nervous systems of a wide range of animals causing alterations such as reproductive disorders and cancer. Detection of these contaminants in water systems is important to protect sensitive environmental sites and reduce the risk of toxins entering the food chain. A modular platform for monitoring toxins in water and food production facilities, using biosensors derived from aquatic organisms, was the main goal of RADAR Project.

Self-propelled continuous-flow PCR in capillary-driven microfluidic device: Microfluidic behavior and DNA amplification

Continuous-flow polymerase chain reaction (CF-PCR) in microfluidic devices has a great potential for on-site detection of various pathogens and food species because of their high speed deoxyribo nucleic acid (DNA) amplification. However, flow-controlled pumps such as syringe pumps are absolutely necessary, which cause complex and difficult operations. Here, we present a self-propelled CF-PCR (SP-CF-PCR) in a microfluidic device which requires no external pumps to control the flow. The PCR solution is simply dropped onto the inlet and is autonomously transported by capillary forces. One of the difficulties in addressing the capillary flow in PCR microfluidic device is that the temperature of the PCR solution is periodically switched. Differing from previous theoretical approaches which deal with single steady-state temperature zone, for the first time, the displacement of capillary flow during temperature switching was mathematically formulated and was simulated by using experimental values of viscosities and capillary pressures at each temperature as parameters for simulation. Basing on the excellent matching between simulated and experimental data of the capillary flow, we were able to reveal the optimized design of 150-μm-wide and 150-μm-deep microchannel that successfully transported over 1600mm for carrying out PCR within less than 14min solely by capillary forces. We also demonstrated the SP-CF-PCR for verifying our concept. The specific amplifications of 295bp of β-actin from human genome, 232bp of AH1pdm influenza virus and 95bp of 16S rDNA of Escherichia coli genomic DNA were successfully achieved, proving the application potentials of our device.

Magnetic nanoparticles as new diagnostic tools in medicine

Magnetic iron oxide nanoparticles have raised much interest during the recent years due to their novel properties (superparamagnetism, high saturation field, blocking temperature, etc.) and potential applications in organic synthesis, biotechnology and finally in medicine. The medicinal applications include: controlled drug delivery systems (DDS), magnetic resonance imaging (MRI), magnetic fluid hyperthermia (MFH), macromolecules and pathogens separation, cancer therapy and so on. In this paper we would like to present the newest literature reports concerning usage of MNPs in medicinal diagnostics such as: - magnetic separations of DNA (immobilization, isolation, diagnosis of genetic disorders and detection of exogenous substances in the organisms) - magnetic immobilization of proteins (applications in biotechnology, medicine, and catalysis) - magnetic separations of pathogens (i.e. isolation of bacteria, detection of various pathogens) - magnetic resonance imaging (imaging contrast agents, lymphangiography).

Biosensors as Novel Platforms for Detection of Food Pathogens and Allergens

Food safety involves preparation and storage of food in different ways to prevent food-borne illness. Ever increasing incidences of food-borne diseases have led food industry to employ rapid and inexpensive method of analysis. It is important not only for health, but also from economic point of view because food-borne diseases result in financial losses. Stringent laws have been made for preparation, packaging, and storage of food. Various techniques are used in food industry but biosensing has received considerable attention due to its high specificity and quick response at low cost. The present review describes use of biosensors for the detection of various pathogens, allergens, pesticide residues, natural contaminants, and other toxic substances in food. Different types of biosensors are used to serve the purpose of food quality assurance such as electrochemical, optical, piezoelectric, and thermometric. Various materials such as monoclonal antibodies, aptamers, RNA, and DNA have been used to enhance the sensitivity and specificity of biosensors for food. Employment of nanoparticles in fabrication of biosensors has shown remarkable potential because of their unique properties at small scale. A wide variety of nanomaterials such as carbon nanotubes, nanoparticles, nanowires, and quantum dots have been used for fabrication of biosensors. Attempts have been made to achieve real-time detection of food contaminants and a little success has been obtained in this regard because more or less, almost every technique requires sample preparation. Highly sensitive and selective biosensors have been developed which showed great potential to mark the presence of food contaminants close to real-time detection. Sensing strategies are moving towards the perfection to obtain real-time detection while assuring high quality of food which is free from any type of contamination.

Design of an improved multiplex PCR method for diagnosis of enterohaemoraghic E.coli and enteropathogic E.coli pathotypes.

We aimed to develop a multiplex PCR assay for specific detection of EPEC and EHEC pathotypes based on specific marker genes. About 2.5 million infant's morbidity in developing countries occurs by E.coli pathotypes because of diarrhea and intestinal diseases. The traditional phenotypic methods are time consuming and sometimes detection and differentiation of the pathotypes are not done easily. Multiplex PCR technology is used as a sensitive, specific and rapid molecular method for detection of various pathogens. PCR reactions were performed with primers which targeted the virulence genes selected for each category (stx 1 , stx 2 genes for EHEC and bfpA for EPEC). For preparation of a positive control, the PCR products were cloned in pTZ57R/T plasmid. The same PCR reactions were done but in presence of genomes of various negative control bacteria for evaluation of test specificity. As expected, gel agarose electrophoresis of PCR products of the stx 1 , stx 2 and bfpA, showed 329bp, 586bp and459bp bands respectively. Result of amplification using negative control genomes as template was negative. The multiplex PCR assay followed by capillary electrophoresis presented in the present paper provides a simple, reliable, and rapid procedure that in a single reaction identifies the four main pathotypes of E. coli. This assay will replace the previous molecular genetics methods used in our laboratory and work as an important supplement to the more time consuming phenotypic assays.

Cloning approach and functional analysis of anti-intimin single-chain variable fragment (scFv)

BackgroundIntimin is an important virulence factor involved in the pathogenesis of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC). Both pathogens are still important causes of diarrhea in children and adults in many developing and industrialized countries. Considering the fact that antibodies are important tools in the detection of various pathogens, an anti-intimin IgG2b monoclonal antibody was previously raised in immunized mice with the conserved sequence of the intimin molecule (int388-667). In immunoblotting assays, this monoclonal antibody showed excellent specificity. Despite good performance, the monoclonal antibody failed to detect some EPEC and EHEC isolates harboring variant amino acids within the 338-667 regions of intimin molecules. Consequently, motivated by its use for diagnosis purposes, in this study we aimed to the cloning and expression of the single-chain variable fragment from this monoclonal antibody (scFv).FindingsAnti-intimin hybridoma mRNA was extracted and reversely transcripted to cDNA, and the light and heavy chains of the variable fragment of the antibody were amplified using commercial primers. The amplified chains were cloned into pGEM-T Easy vector. Specific primers were designed and used in an amplification and chain linkage strategy, obtaining the scFv, which in turn was cloned into pAE vector. E. coli BL21(DE3)pLys strain was transformed with pAE scFv-intimin plasmid and subjected to induction of protein expression. Anti-intimin scFv, expressed as inclusion bodies (insoluble fraction), was denatured, purified and submitted to refolding. The protein yield was 1 mg protein per 100 mL of bacterial culture. To test the functionality of the scFv, ELISA and immunofluorescence assays were performed, showing that 275 ng of scFv reacted with 2 mg of purified intimin, resulting in an absorbance of 0.75 at 492 nm. The immunofluorescence assay showed a strong reactivity with EPEC E2348/69.ConclusionThis study demonstrated that the recombinant anti-intimin antibody obtained is able to recognize the conserved region of intimin (Int388-667) in purified form and the EPEC isolate.

The development of protein chip using protein G for the simultaneous detection of various pathogens

A protein chip using protein G for the simultaneous detection of various pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, Yersinia enterocolitica, and Legionella pneumophila was developed. In order to endow the orientation of antibody molecules on solid surface, protein G was introduced. The protein G on gold (Au) surface modified with 11-mercaptoundecanoic acid (MUA) was arrayed and then four different kinds of monoclonal antibodies (Mabs) against pathogens ( E. coli O157:H7, S. typhimurium, Y. enterocolitica, and L. pneumophila) on protein G spots were selectively arrayed using a microarrayer, and its spatial density was over 2400 spots cm 2. Using the constructed protein chip, the various pathogens such as E. coli O157:H7, S. typhimurium, Y. enterocolitica, and L. pneumophila could be detected by a sandwich method and its lowest detection limit for E. coli O157:H7 was 10 2 CFU/ml. The proposed fabrication technique of protein chip for the detection of various pathogens could be applied to construct other protein chips with a high efficiency.

La PCR en temps réel : principe et application en infectiologie

Objectives Real time PCR or quantitative reaction constitutes a rapid, sensitive and specific method for the detection of infective agents. Methods Fluorescence is used to measure the accumulation of products of polymerase chain after each cycle. Measurement of fluorescence allows for direct quantification of target DNA present in the sample. Detection and quantification are determined simultaneously. Limits of detection are usually about a femtogram for purified DNA from pathogenic agents, and about a picogram for DNA from samples containing pathogens. Applications Real time PCR method is already seen as a tool applicable to molecular epidemiology, for detection of emerging pathogens and for antibiotic resistance detection. The objectives of this review include four principal techniques used for real time detection and quantification, with current application of real time PCR for the detection of various pathogens and the development of new tests.

Liposome encapsulation of the internal control for whole process quality assurance of nucleic acid amplification-based assays

A system intended for whole process quality assurance of nucleic acid amplification assays was developed based on the use of liposomes as cell-mimicking vehicles for the internal control, allowing introduction of the internal control directly into the crude biological specimens. By the proof of principle testing, the Roche Cobas Amplicor® CT assay was chosen as model system and the Roche CT/NG Internal Control was thus loaded into the liposomes. The liposome/DNA particles were spiked into a Chlamydia trachomatis-positive urine specimen. A quantitative “in-house” duplex real-time C. trachomatis PCR assay showed that liposomes having Blue Dextran 2000 polysaccharide co-entrapped were the most suited particles as they were efficiently deposited by the centrifugation carried out according to the Roche urine specimen preparation procedure. Furthermore, it was demonstrated that the liposome/DNA particles might be used for whole process quality assurance of Amplicor® assay without major modifications of the assay protocol. An additional feature of the use of these liposomes was that the pellet became blue coloured and that might facilitate a thorough removal of the urine supernatant without increasing the risk of disturbance of the pellet. Principally, the liposome/internal control system is versatile and seems to be applicable for whole process quality control of amplification-based assays for detection of various pathogens.