Platform For Pathogen Detection Research Articles

PHDtools: A platform for pathogen detection and multi-dimensional genetic signatures decoding to realize pathogen genomics data analyses online

ObjectivesFacing the emerging diseases, rapid identification of the pathogen and multi-dimensional characterization of the genomic features at both isolate-level and population-level through high-throughput sequencing data can provide invaluable information to guide the development of antiviral agents and strategies. However, a user-friendly program is in urgent need for clinical laboratories without bioinformatics background to decode the complex big genomics data. MethodsIn this study, we developed an interactive online platform named PHDtools with a total of 15 functions to analyze metagenomics data to identify the potential pathogen and decode multi-dimensional genetic signatures including intra-/inter-host variations and lineage-level variations. The platform was applied to analyze the meta-genomic data of the samples collected from the 172 imported COVID-19 cases. ResultsAccording to the analytical results of mNGS, 27 patients were found to have the co-infections of SARS-CoV-2 with either influenza virus (n = 9) or human picobirnavirus (n = 19). Enough coverages of all the assembled SARS-CoV-2 genomes provided the sub-lineages of Omicron variant, and the number of mutations in the non-structural genes and M gene was increased, as well as the intra-host variations occurred in E and M gene were under positive selection (Ka/Ks > 1). These findings of increased or changed mutations in the SARS-CoV-2 genome characterized the current adaptive evolution patterns of Omicron sub-lineages, and revealed the evolution speed of these sub-lineages might increase. ConclusionsConsequently, the application of PHDtools has proved that this platform is accurate, user-friendly and convenient for clinical users who are deficient in bioinformatics, and the clinical monitor of SARS-CoV-2 genomes by PHDtools also highlighted the potential evolution features of current SARS-CoV-2 and indicated that the development of anti-SARS-CoV-2 agents and new-designed vaccines should incorporate the gene variations other than S gene.

A LAMP-based hydrogen ion selective electrochemical sensor for highly sensitive detection of Mycoplasma pneumoniae.

A concise and rapid detection method for Mycoplasma pneumoniae is urgently required due to its severe impact on human health. To meet such a need, this study proposed and constructed an innovative point-of-care testing (POCT) platform that consists of a hydrogen ion-selective loop-mediated isothermal amplification (H+-LAMP) sensor and an electrochemical detection device. The H+-LAMP sensor successfully integrated the working and reference electrodes and converted the H+ generated during the LAMP process into an electrochemical signal. High sensitivity and stability for pathogen detection were also achieved by treating the working electrode with an electrodeposited polyaniline solid contact layer and by using an ion-selective membrane. As a result, the sensor shows a sensitivity of 68.26 mV per pH, a response time of less than 2 s, and a potential drift of less than 5 mV within one hour, which well meets the urgent need. The results also demonstrated that the detection limit for Mycoplasma pneumoniae was lowered to 1 copy per μL, the nucleic acid extraction and detection process could be completed in 30 minutes, and the impact of interfering ions on the sensor was negligible. Validation with 20 clinical samples yielded satisfactory results. More importantly, the storage lifespan of such an electrochemical sensor is over seven days, which is a great advantage for on-site pathogen detection. Therefore, the hydrogen ion-selective sensor constructed in this investigation is particularly suitable as a core component for instant pathogen detection platforms.

Loop-Mediated Isothermal Amplification-Integrated CRISPR Methods for Infectious Disease Diagnosis at Point of Care.

Infectious diseases continue to pose an imminent threat to global public health, leading to high numbers of deaths every year and disproportionately impacting developing countries where access to healthcare is limited. Biological, environmental, and social phenomena, including climate change, globalization, increased population density, and social inequity, contribute to the emergence of novel communicable diseases. Rapid and accurate diagnoses of infectious diseases are essential to preventing the transmission of infectious diseases. Although some commonly used diagnostic technologies provide highly sensitive and specific measurements, limitations including the requirement for complex equipment/infrastructure and refrigeration, the need for trained personnel, long sample processing times, and high cost remain unresolved. To ensure global access to affordable diagnostic methods, loop-mediated isothermal amplification (LAMP) integrated clustered regularly interspaced short palindromic repeat (CRISPR) based pathogen detection has emerged as a promising technology. Here, LAMP-integrated CRISPR-based nucleic acid detection methods are discussed in point-of-care (PoC) pathogen detection platforms, and current limitations and future directions are also identified.

Whole-genome detection using multivalent DNA-coated colloids

To minimize the incorrect use of antibiotics, there is a great need for rapid and inexpensive tests to identify the pathogens that cause an infection. The gold standard of pathogen identification is based on the recognition of DNA sequences that are unique for a given pathogen. Here, we propose and test a strategy to develop simple, fast, and highly sensitive biosensors that make use of multivalency. Our approach uses DNA-functionalized polystyrene colloids that distinguish pathogens on the basis of the frequency of selected short DNA sequences in their genome. Importantly, our method uses entire genomes and does not require nucleic acid amplification. Polystyrene colloids grafted with specially designed surface DNA probes can bind cooperatively to frequently repeated sequences along the entire genome of the target bacteria, resulting in the formation of large and easily detectable colloidal aggregates. Our detection strategy allows "mix and read" detection of the target analyte; it is robust and highly sensitive over a wide concentration range covering, in the case of our test target genome Escherichia coli bl21-de3, 10 orders of magnitude from [Formula: see text] to [Formula: see text] copies/mL. The sensitivity compares well with state-of-the-art sensing techniques and has excellent specificity against nontarget bacteria. When applied to real samples, the proposed technique shows an excellent recovery rate. Our detection strategy opens the way to developing a robust platform for pathogen detection in the fields of food safety, disease control, and environmental monitoring.

Development and evaluation of Microbe Finder (MiFi)®: a novel in silico diagnostic platform for pathogen detection from metagenomic data.

Introduction. With expanding demand for diagnostics, newer methodologies are needed for faster, user-friendly and multiplexed pathogen detection. Metagenome-based diagnostics offer potential solutions to address these needs as sequencing technologies have become affordable. However, the diagnostic utility of sequencing technologies is currently limited since analysis of the large amounts of data generated, are either computationally expensive or carry lower sensitivity and specificity for pathogen detection.Hypothesis/Gap Statement. There is a need for novel, user friendly, and computationally inexpensive platforms for metagenome sequence analysis for diagnostic applications.Methods. In this study, we report the use of MiFi® (Microbe Finder), a computationally inexpensive algorithm with a user-friendly online interface, for accurate, rapid and multiplexed pathogen detection from metagenome sequence data. Detection is accomplished based on identification of signature genomic sequence segments of the target pathogen in metagenome sequence data. In this study we used bovine respiratory disease (BRD) complex as a model.Results and Conclusions. Using MiFi®, multiple target bacteria and a DNA virus were successfully detected in a multiplex format from metagenome sequences acquired from bovine lung tissue. Overall, 51 clinical samples were assessed and MiFi® showed 100 % analytical specificity and varying levels of analytical sensitivity (62.5 %-100 %) when compared with other traditional pathogen detection techniques, such as PCR. Consistent detection of bacteria was possible from lung samples artificially spiked with 109-104 c.f.u. of Mannheimia haemolytica.

Computational approaches screening DNA aptamers against conserved outer membrane protein W of Vibrio cholerae O1- an investigation expanding the potential for point-of-care detection with aptasensors

Foodborne outbreaks urge public health domain to upgrade diagnosis by means of simpler, quicker, and more affordable pathogen detection methods. A molecular recognition probe against an analyte of interest makes up a biosensor, along with a method for turning the recognition event into a quantifiable signal. Single-stranded DNA or RNA aptamers are promising bio-recognition molecules for a range of targets, including a wide range of non-nucleic acid targets with which they are highly specific and affine. In the proposed study, 40 DNA aptamers were screened and analyzed interactions using in-silico SELEX procedures, which can selectively interact with active sites at the extracellular region of the Outer membrane Protein W (OmpW) of Vibrio Cholerae. Multiple modeling techniques, like protein structural prediction with I-TASSER, aptamer structural modeling using M-fold, RNA composer, protein-DNA docking using HADDOCK, and large-scale (500 ns) molecular dynamics simulations through GROMACS have been employed. Out of 40, six aptamers having lowest free energy were docked against the predicted active site at the extracellular region of OmpW. VBAPT4-OmpW and VBAPT17-OmpW, the two highest-scoring Aptamer-Protein complexes, were chosen for molecular dynamics simulations. VBAPT4-OmpW is quite unable to attain its structural local minima after 500 ns. But VBAPT17-OmpW is showing great stability and is not destructive even after 500 ns. RMSF, DSSP, PCA, and Essential Dynamics all provided additional confirmation. Current findings, combined with the fabrication of biosensor devices, could pave the way for an innovative pathogen detection platform with high sensitivity, along with an effective and low-impact curative strategy for corresponding diseases.Communicated by Ramaswamy H. Sarma

CRISPR-Cas- and Aptamer-based Systems for Diagnosing Pathogens: A Review

Pathogenic infections cause severe clinical illnesses in humans and animals. Increased encounters between humans and animals and constant environmental changes exacerbate the transmission of zoonotic infectious diseases. Recently, the World Health Organization has declared some zoonotic epidemics as public health emergencies of international concern. Hence, rapid and accurate detection of the causative pathogen is particularly essential in combating emerging and re-emerging infectious diseases. Traditional pathogen detection tools are time-consuming, costly, and require skilled personnel, which greatly hinder the development of rapid diagnostic tests, particularly in resource-constrained regions. Clustered regularly interspaced short palindromic repeats (CRISPR-)-Cas- and aptamer-based platforms have replaced traditional pathogen detection methods. Herein we review two novel next-generation core pathogen detection platforms that are utilized for clinical and foodborne pathogenic microorganisms: CRISPR-Cas-based systems, including dCas9, Cas12a/b, Cas13, and Cas14; and aptamer-based biosensor detection tools. We highlight CRISPR-Cas- and aptamer-based techniques and compare the strengths and weaknesses. CRISPR-Cas-based tools require cumbersome procedures, such as nucleic acid amplification and extraction, while aptamer-based tools require improved sensitivity. We review the combination of CRISPR-Cas- and aptamer-based techniques as a promising approach to overcome these deficiencies. Finally, we discuss Cas14-based tools as functionally stronger platforms for the detection of non-nucleic acid targets.

A phage-based magnetic relaxation switching biosensor using bioorthogonal reaction signal amplification for Salmonella detection in foods

Phages are uniquely suited for bacterial detection due to their low cost and ability to recognize live bacteria. Herein, our work establishes the proof-of-concept detection of Salmonella in orange juice based on a phage-mediated portable magnetic relaxation switching (MRS) biosensor. The limit of quantification (LOQ) could reach 5 CFU/mL (95 % confidence interval [CI]: 4–7, N = 4) with a linear range of 102-108 CFU/mL, which has improved 10-fold than that without bioorthogonal signal amplification. The recovery rate of the phage-based MRS biosensor was 95.0 % (95 % confidence interval [CI]: 89.0 %-100.9 %, N = 6). The specificity of the phage-based MRS biosensor was 100 % without false-positive results. In addition, this sensor was able to detect <10 CFU per 25 mL of Salmonella in orange juice with 4-h pre-enrichment. The result from the phage-based MRS biosensor is consistent with that from the standard plate count method. This sensor provides a reliable and ultrasensitive detection platform for pathogens.

Phage-based technologies for highly sensitive luminescent detection of foodborne pathogens and microbial toxins: A review.

Foodborne pathogens and microbial toxins are the main causes of foodborne illness. However, trace pathogens and toxins in foods are difficult to detect. Thus, techniques for their rapid and sensitive identification and quantification are urgently needed. Phages can specifically recognize and adhere to certain species of microbes or toxins due to molecular complementation between capsid proteins of phages and receptors on the host cell wall or toxins, and thus they have been successfully developed into a detection platform for pathogens and toxins. This review presents an update on phage-based luminescent detection technologies as well as their working principles and characteristics. Based on phage display techniques of temperate phages, reporter gene detection assays have been designed to sensitively detect trace pathogens by luminous intensity. By the host-specific lytic effects of virulent phages, enzyme-catalyzed chemiluminescent detection technologies for pathogens have been exploited. Notably, these phage-based luminescent detection technologies can discriminate viable versus dead microbes. Further, highly selective and sensitive immune-based assays have been developed to detect trace toxins qualitatively and quantitatively via antibody analogs displayed by phages, such as phage-ELISA (enzyme-linked immunosorbent assay) and phage-IPCR (immuno-polymerase chain reaction). This literature research may lead to novel and innocuous phage-based rapid detection technologies to ensure food safety.

Evaluations of Clinical Utilization of Metagenomic Next-Generation Sequencing in Adults With Fever of Unknown Origin.

IntroductionThe diagnosis of infection-caused fever of unknown origin (FUO) is still challenging, making it difficult for physicians to provide an early effective therapy. Therefore, a novel pathogen detection platform is needed. Metagenomic next-generation sequencing (mNGS) provides an unbiased, comprehensive technique for the sequence-based identification of pathogenic microbes, but the study of the diagnostic values of mNGS in FUO is still limited.MethodsIn a single-center retrospective cohort study, 175 FUO patients were enrolled, and clinical data were recorded and analyzed to compare mNGS with culture or traditional methods including as smears, serological tests, and nucleic acid amplification testing (NAAT) (traditional PCR, Xpert MTB/RIF, and Xpert MTB/RIF Ultra).ResultsThe blood mNGS could increase the overall rate of new organisms detected in infection-caused FUO by roughly 22.9% and 19.79% in comparison to culture (22/96 vs. 0/96; OR, ∞; p = 0.000) and conventional methods (19/96 vs. 3/96; OR, 6.333; p = 0.001), respectively. Bloodstream infection was among the largest group of those identified, and the blood mNGS could have a 38% improvement in the diagnosis rate compared to culture (19/50 vs. 0/50; OR, ∞; p = 0.000) and 32.0% compared to conventional methods (16/50 vs. 3/50; OR, 5.333; p = 0.004). Among the non-blood samples in infection-caused FUO, we observed that the overall diagnostic performance of mNGS in infectious disease was better than that of conventional methods by 20% (9/45 vs. 2/45; OR, 4.5; p = 0.065), and expectedly, the use of non-blood mNGS in non-bloodstream infection increased the diagnostic rate by 26.2% (8/32 vs. 0/32; OR, ∞; p = 0.008). According to 175 patients’ clinical decision-making, we found that the use of blood mNGS as the first-line investigation could effectively increase 10.9% of diagnosis rate of FUO compared to culture, and the strategy that the mNGS of suspected parts as the second-line test could further benefit infectious patients, improving the diagnosis rate of concurrent infection by 66.7% and 12.5% in non-bloodstream infection, respectively.ConclusionThe application of mNGS in the FUO had significantly higher diagnostic efficacy than culture or other conventional methods. In infection-caused FUO patients, application of blood mNGS as the first-line investigation and identification of samples from suspected infection sites as the second-line test could enhance the overall FUO diagnosis rate and serve as a promising optimized diagnostic protocol in the future.

Bio-SCAN: A CRISPR/dCas9-Based Lateral Flow Assay for Rapid, Specific, and Sensitive Detection of SARS-CoV-2.

Simple, rapid, specific, and sensitive point-of-care detection methods are needed to contain the spread of SARS-CoV-2. CRISPR/Cas9-based lateral flow assays are emerging as a powerful alternative for COVID-19 diagnostics. Here, we developed Bio-SCAN (biotin-coupled specific CRISPR-based assay for nucleic acid detection) as an accurate pathogen detection platform that requires no sophisticated equipment or technical expertise. Bio-SCAN detects the SARS-CoV-2 genome in less than 1 h from sample collection to result. In the first step, the target nucleic acid sequence is isothermally amplified in 15 min via recombinase polymerase amplification before being precisely detected by biotin-labeled nuclease-dead SpCas9 (dCas9) on commercially available lateral flow strips. The resulting readout is visible to the naked eye. Compared to other CRISPR-Cas-based pathogen detection assays, Bio-SCAN requires no additional reporters, probes, enhancers, reagents, or sophisticated devices to interpret the results. Bio-SCAN is highly sensitive and successfully detected a clinically relevant level (4 copies/μL) of synthetic SARS-CoV-2 RNA genome. Similarly, Bio-SCAN showed 100% negative and 96% positive predictive agreement with RT-qPCR results when using clinical samples (86 nasopharyngeal swab samples). Furthermore, incorporating variant-specific sgRNAs in the detection reaction allowed Bio-SCAN to efficiently distinguish between the α, β, and δ SARS-CoV-2 variants. Also, our results confirmed that the Bio-SCAN reagents have a long shelf life and can be assembled locally in nonlaboratory and limited-resource settings. Furthermore, the Bio-SCAN platform is compatible with the nucleic acid quick extraction protocol. Our results highlight the potential of Bio-SCAN as a promising point-of-care diagnostic platform that can facilitate low-cost mass screening for SARS-CoV-2.

A Novel Rapid Test to Detect Anti-SARS-CoV-2 N Protein IgG Based on Shear Horizontal Surface Acoustic Wave (SH-SAW).

Since the Coronavirus disease 2019 (COVID-19) pandemic outbreak, many methods have been used to detect antigens or antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including viral culture, nucleic acid test, and immunoassay. The shear-horizontal surface acoustic wave (SH-SAW) biosensor is a novel pathogen detection platform with the advantages of high sensitivity and short detection time. The objective of this study is to develop a SH-SAW biosensor to detect the anti-SARS-CoV-2 nucleocapsid antibody. The rabbit sera collected from rabbits on different days after SARS-CoV-2 N protein injection were evaluated by SH-SAW biosensor and enzyme-linked immunosorbent assay (ELISA). The results showed that the SH-SAW biosensor achieved a high correlation coefficient (R = 0.9997) with different concentrations (34.375–1100 ng/mL) of the “spike-in” anti-N protein antibodies. Compared to ELISA, the SH-SAW biosensor has better sensitivity and can detect anti-N protein IgG signals earlier than ELISA on day 6 (p < 0.05). Overall, in this study, we demonstrated that the SH-SAW biosensor is a promising platform for rapid in vitro diagnostic (IVD) testing, especially for antigen or antibody testing.

Direct Transverse Relaxation Time Biosensing Strategy for Detecting Foodborne Pathogens through Enzyme-Mediated Sol-Gel Transition of Hydrogels.

In this work, we develop a direct transverse relaxation time (T2) biosensing strategy and employ it for assaying foodborne pathogens relying on the alkaline phosphatase (ALP)-mediated sol-gel transition of hydrogels. ALP can catalyze the reaction to generate an acidic environment to transform the sol-state alginate solution to hydrogel, and this hydrogelation process can directly regulate the diffusion rate of water protons that results in a T2 change of water molecules. By means of enzyme-modulated sol-gel transition and antigen-antibody interactions, this T2 biosensor displays high sensitivity for detecting 50 CFU/mL S. enteritidis within 2 h. This biosensing strategy directly modulates the water molecules rather than magnetic probes in traditional methods, offering a straightforward, novel, and sensitive platform for pathogen detection.

Rapid and sensitive pathogen detection platform based on a lanthanide-labeled immunochromatographic strip test combined with immunomagnetic separation

Sensitive point-of-care pathogen detection methods are urgently required as pathogens pose a significant threat to food safety and human health. Here, we reported a platform based on a lanthanide-labeled immunochromatographic strip test combined with immunomagnetic separation for the detection of pathogens in food and clinical samples. First, immunomagnetic separation technology was employed as a pre-treatment to eliminate the effect of background in the samples. Then, the lanthanide chelate-loaded carboxyl-modified polystyrene nanoparticles, exhibiting bright luminescence, wide Stokes shift, and excellent stability, were prepared and used to label pathogens with antibody. Finally, an immunochromatographic strip test, based on a sandwich format and supported by our custom-designed portable reader, was used to rapidly quantify the result. Using Salmonella typhimurium as a model pathogen, under optimal conditions, we achieved a detection limit of 103 CFU/mL and a linear range of 104–106 CFU/mL in milk and human serum with good recovery, reproducibility, and specificity. Compared with the existing colloidal gold-based immunochromatographic strip test, our platform showed high sensitivity (100-fold improvement) and provided quantitative determination. Our detection platform was therefore demonstrated to be highly sensitive, rapid (within 1.5 h), and cost-effective, providing a promising platform for point-of-care pathogen detection in food and clinical fields.

One-step and DNA amplification-free detection of Listeria monocytogenes in ham samples: Combining magnetic relaxation switching and DNA hybridization reaction

Early screening of L. monocytogenes in ready-to-eat food can prevent and control its harmful effects. In this study, we propose a highly sensitive magnetic DNA sensor based on nucleic acid hybridization reaction and magnetic signal readout. We design the L. monocytogenes specific probe1 and probe2 and label them on the 30 and 250 nm magnetic nanoparticles, respectively. The hybridization reaction between the magnetic probes and DNA of L. monocytogenes could form a sandwich nanocomplex. After magnetic separation, the unbound MNP30–probe2 can act as the transverse relaxation time (T2) signal readout probe. This assay allows the one-step detection of L. monocytogenes as low as 50 CFU/mL within 2 h without DNA amplification, and the average recovery in the spiked ham sausage samples can reach 92.6%. This system integrates the high sensitivity of magnetic sensing and high efficiency of hybridization reaction, providing a promising detection platform for pathogens.

657. Evaluation of Nanopore-Based 16S Ribosomal RNA (rRNA) Gene Sequencing for the Development of a Rapid Infection Intervention Clinical Service

BackgroundRapid and accurate identification of bacteria is the basis of appropriate antibiotic treatment and effective clinical decision-making. Next-generation sequencing (NGS) platforms such as Oxford Nanopore Technologies (ONT) holds the promise of a diagnostic revolution by overcoming the limitations of culture-based identification with rapid molecular detection of bacteria. We have developed a pilot to evaluate an ONT 16S rRNA gene assay with the ability to provide real-time analysis and identification of bacterial species. Our aim was to investigate whether long-read sequencing and high-speed analysis can be combined to create a clinically useful, rapid diagnostic tool.MethodsA collection of bacterial isolates representing pathogenic species received by the clinical laboratory over 1 year was assembled. Sample preparation was as described in the ONT 16S protocol and included bead beating sample disruption, MagNA Pure automated nucleic acid extraction (Roche), and PCR amplification (Thermo). Sequencing was performed on the MinION and GridION X5 platforms. Output was analyzed with ONT’s automated EPI2ME 16S pipeline which assigns reads to taxa using BLAST results and the NCBI 16S Bacterial database.ResultsA total of 155 clinical samples with 139 species were sequenced. 119 species were identified at the species level. For 20 samples, a species in the same genus claimed the majority of reads, with the true species being matched to 3%-41% of reads. The average proportion of reads assigned to the correct species was 62.2%, specifically 67% for non-Enterobacteriaceae and 33% for Enterobacteriaceae. 4 clinical samples (3 Bronchoalveolar lavages (BALs), positive for (1) K. pneumoniae, (2) S. pneumoniae, and (3) S. pneumoniae, S. enterica, and S. typhimurium, and 1 bone positive for P. aeruginosa) were also analyzed with sequencing results matching culture.ConclusionEarly results show that 16S rRNA sequencing coupled with real-time analysis was able to accelerate pathogen detection and was able to discriminate the majority of species from a relevant clinical collection. Pipeline refinement is required and subsequent confirmatory consensus-based identification may be a helpful adjunct. Nanopore sequencing shows promise as a rapid bacterial pathogen detection platform for clinical service.Disclosures All authors: No reported disclosures.

A Rapid and Low-Cost Pathogen Detection Platform by Using a Molecular Agglutination Assay.

Rapid and low-cost pathogen diagnostic approaches are critical for clinical decision-making procedures. Cultivating bacteria often takes days to identify pathogens and provide antimicrobial susceptibilities. The delay in diagnosis may result in compromised treatment and inappropriate antibiotic use. Over the past decades, molecular-based techniques have significantly shortened pathogen identification turnaround time with high accuracy. However, these assays often use complex fluorescent labeling and nucleic acid amplification processes, which limit their use in resource-limited settings. In this work, we demonstrate a wash-free molecular agglutination assay with a straightforward mixing and incubation step that significantly simplifies procedures of molecular testing. By targeting the 16S rRNA gene of pathogens, we perform a rapid pathogen identification within 30 min on a dark-field imaging microfluidic cytometry platform. The dark-field images with low background noise can be obtained using a narrow beam scanning technique with off-the-shelf complementary metal oxide semiconductor (CMOS) imagers such as smartphone cameras. We utilize a machine learning algorithm to deconvolute topological features of agglutinated clusters and thus quantify the abundance of bacteria. Consequently, we unambiguously distinguish Escherichia coli positive from other E. coli negative among 50 clinical urinary tract infection samples with 96% sensitivity and 100% specificity. Furthermore, we also apply this quantitative detection approach to achieve rapid antimicrobial susceptibility testing within 3 h. This work exhibits easy-to-use protocols, high sensitivity, and short turnaround time for point-of-care testing uses.

Loop-mediated isothermal amplification (LAMP): A novel rapid detection platform for pathogens.

Foodborne bacterial infections and diseases have been considered to be a major threat for public health in the worldwide. Increased incidence of human diseases caused by foodborne pathogens have been correlated with growing world population and mobility. Loop-mediated isothermal amplification (LAMP) has been regarded as an innovative gene amplification technology and emerged as an alternative to PCR-based methodologies in both clinical laboratory and food safety testing. Nowadays, LAMP has been applied to detection and identification on pathogens from microbial diseases, as it showed significant advantage in high sensitivity, specificity and rapidity. The high sensitivity of LAMP enables detection of the pathogens in sample materials even without time consuming sample preparation. An overview of LAMP mainly containing the development history, reaction principle and its application to four kind of foodborne pathogens detection are presented in this paper. As concluded, with the advantages of rapidity, simplicity, sensitivity, specificity and robustness, LAMP is capable of applications for clinical diagnosis as well as surveillance of infection diseases. Moreover, the main purpose of this paper is to provide theoretical basis for the clinical application of LAMP technology.

Plasma micro-nanotextured polymeric micromixer for DNA purification with high efficiency and dynamic range

We present a polymeric microfluidic chip capable of purifying DNA through solid phase extraction. It is designed to be used as a module of an integrated Lab-on-chip platform for pathogen detection, but it can also be used as a stand-alone device. The microfluidic channels are oxygen plasma micro-nanotextured, i.e. randomly roughened in the micro-nano scale, a process creating high surface area as well as high density of carboxyl groups (COOH). The COOH groups together with a buffer that contains polyethylene glycol (PEG), NaCl and ethanol are able to bind DNA on the microchannel surface. The chip design incorporates a mixer so that sample and buffer can be efficiently mixed on chip under continuous flow. DNA is subsequently eluted in water. The chip is able to isolate DNA with high recovery efficiency (96± 11%) in an extremely large dynamic range of prepurified Salmonella DNA as well as from Salmonella cell lysates that correspond to a range of 5 to 1.9 × 108 cells (0.263 fg to 2 × 500 ng). The chip was evaluated via absorbance measurements, polymerase chain reaction (PCR), and gel electrophoresis.

A highly sensitive and flexible magnetic nanoprobe labeled immunochromatographic assay platform for pathogen Vibrio parahaemolyticus

A magnetic nanoprobe labeled immunochromatographic test strip (MNP/ICTS) was developed to detect food-borne pathogen Vibrio parahaemolyticus. Specific antibody against V. parahaemolyticus was used as test line by coating onto the nitrocellulose membrane. Magnetic nanoprobe was prepared by immobilizing the specific antibody onto the surface of superparamagnetic nanoparticles. Specificity and sensitivity of the MNP/ICTS system were verified by artificially contaminated shrimp homogenate samples. Reliability and application feasibility of the MNP/ICTS system were demonstrated by using seafood samples (n=36). Comparing with polymerase chain reaction (PCR) and traditional culture methods, the MNP/ICTS system is found to be not only a rapid qualitative analysis (~10min), but also an accurately quantitative detection platform. Through its rapid magnetic separation property, the MNP/ICTS system is capable to flexibly combine with a sample enrichment and pre-incubation process. This combination makes the qualitative sensitivity for the food samples surged more than 100-fold. A naked-eye observation of 1.58×102CFU/g V. parahaemolyticus was realized. This sensitivity could meet the V. parahaemolyticus test threshold value in many countries. Also, the total sample pre-treatment plus MNP/ICTS assay only needs about 4.5h. Namely, we can get test results in a day. Hence, the developed MNP/ICTS assay platform is simple, rapid and highly sensitive. It is a flexible test platform for pathogen detection. The favorable comparison with PCR and culture methods further proves that the developed MNP/ICTS is applicable into food-borne pathogen or other areas where a simple, rapid, sensitive and point-of-care analysis is desirable.