Pathogen Screening Research Articles

Fluorescent/electrochemical dual-signal response biosensing strategy mediated by DNAzyme-ferrocene-triggered click chemistry for simultaneous rapid screening and quantitative detection of Vibrio parahaemolyticus

To ensure the safety of food, rapid screening and quantitative detection of foodborne pathogens are both significantly required. In this work, a dual-signal response biosensing strategy integrated with the visual fluorescent signal and the quantitative electrochemical signal was developed to simultaneously satisfy these demands. The deoxyribozyme (DNAzyme)-ferrocene immobilized on the MXene/gold nanobipyramid/antimicrobial peptide was employed as the signal probe for dual signal output. In the presence of Vibrio parahaemolyticus (V.P, as the model analyte), the typical immunocomplexes between the signal probe, target, and capture antibody were formed on the sensing electrode. The DNAzyme with excellent catalytic ability allowed the click reaction of 3-azide-7-hydroxycoumarin and 3-butyn-1-ol with the assistance of copper ion (Cu2+) to provide the visual fluorescent signal for rapid screening. Then the ferrocene provided a quantified electrochemical detection signal to further confirm the foodborne pathogen contamination. Notably, the two signals provided a built-in cross-reference correction, which greatly improved the detection accuracy. Under optimum conditions, the proposed biosensing strategy exhibited a wide detectable range of 10–108 CFU·mL−1 and a low detection limit of 6 CFU·mL−1. This dual-signal biosensing strategy provides a useful route to develop simple and novel strategies for the on-site detection of foodborne pathogens.

An achromatic colorimetric nanosensor for sensitive multiple pathogen detection by coupling plasmonic nanoparticles with magnetic separation

Rapid screening of multiple pathogens will greatly improve the efficiency of pandemic prevention and control. Colorimetric methods exhibit the advantages of convenience, portability, low cost, time efficiency, and free of sophisticated instruments, yet usually have difficulties in simultaneous detection and suffer from monotonous color changes with low visual resolution and sensitivity. Hence, coupled three kinds of plasmonic nanoparticles (NPs) with magnetic separation, we developed an achromatic colorimetric nanosensor with highly enhanced visual resolution for simultaneous detection of SARS-CoV-2, Staphylococcus aureus, and Salmonella typhimurium. The achromatic nanosensor was composed of SARS-CoV-2-targeting red gold NPs, S. aureus-targeting yellow silver NPs and S. typhimurium-targeting blue silver triangle NPs mixed as black color. In the detection, three corresponding magnetic probes were added into the above mixture. In the presence of a target pathogen, it would be recognized and combined with corresponding colored reporters and magnetic probes to form sandwich complexes, which were removed by magnetic separation, and the sensor changed from black to a chromatic color (the color of the reporters remained in supernatant). Consequently, different target pathogen induced different color. For example, SARS-CoV-2, S. aureus, and S. typhimurium respectively produced green, purple, and orange. While coexistence of S. aureus and S. typhimurium produced red, and coexistence of S. aureus and SARS-CoV-2 produced blue, etc. Therefore, by observing the color change or measuring the absorption spectra, multiple pathogen detection was achieved conveniently. Compared with most colorimetric sensors, this achromatic nanosensor involved rich color change, thus significantly enhancing visual resolution and inspection sensitivity. Therefore, this sensor opened a promising avenue for efficient monitoring and early warning of food safety and quality.

Antibiogram Screening of Bacterial Pathogens Obtained From Respiratory ‎Manifested Patients in Great Cairo ‎

Respiratory tract infections are the most prevalent infections and implemented in hundred thousand deaths among humans globally, especially in the last years. The goal of the current investigation was to determine the prevalence and antibiotic sensitivity pattern of microorganisms isolated from respiratory samples obtained from laboratories in Great Cairo between January to June 2022. A total of 107 samples distributed as fifteen naso-oral, forty three throat and forty nine sputum were collected. Among them, 91 samples (85.04%) exhibited significant microbial growth on culture media and near equal rate in patients with male and female gender (46 and 45 respectively). The most susceptible ages were both under 12 and over 50 years. The microorganism identification and antibiotic susceptibility testing were conducted. Out of 91isolates species, 56 (61.54%) were Gram-negative bacteria, 35 (38.46%) were Gram-positive bacteria. Prime microorganisms isolated were Klebsiella pneumonia 28 (30.77%), Streptococcous pneumonia 26 (28.57%), Staphylococcous aureus 9 (9.9%), Pseudomonas aeruginosa 8 (8.79%), Escherichia coli (E. coli) 8 (8.79%), Citrobacter spp. 7 (7.69%), Proteus vulgaris 4 (4.4%) and one sample positive for non-lactose fermenting (NLF) E. coli (1.1%). In antimicrobial susceptibility assay, the isolates were mostly resistant to many antibiotics; particularly amoxicillin-clavulanic acid, ampicillin, ceftazidime, clindamycin, oxicillin and trimethoprim-sulfamethoxazole. On the other hand the isolates exposed maximum susceptibility to imipenem and piperacillin-tazobactam (100%), as well as cefoperazone-sulbactam (96.66%) and amikacin (85%).In conclusion, the precise identification of causative microorganisms and their susceptibility profiles to antibiotics is pivotal for selecting targeted and effective antibiotic therapy in respiratory tract infections, and to hinder the emergence of multidrug-resistant bacteria. Gram-negative bacteria exhibited the species most commonly obtained. A high rate of drug resistance was observed in this investigation which may be of public health significance.

Clasnip: a web-based intraspecies classifier and multi-locus sequence typing for pathogenic microorganisms using fragmented sequences.

Bioinformatic approaches for the identification of microorganisms have evolved rapidly, but existing methods are time-consuming, complicated or expensive for massive screening of pathogens and their non-pathogenic relatives. Also, bioinformatic classifiers usually lack automatically generated performance statistics for specific databases. To address this problem, we developed Clasnip (www.clasnip.com), an easy-to-use web-based platform for the classification and similarity evaluation of closely related microorganisms at interspecies and intraspecies levels. Clasnip mainly consists of two modules: database building and sample classification. In database building, labeled nucleotide sequences are mapped to a reference sequence, and then single nucleotide polymorphisms (SNPs) statistics are generated. A probability model of SNPs and classification groups is built using Hidden Markov Models and solved using the maximum likelihood method. Database performance is estimated using three replicates of two-fold cross-validation. Sensitivity (recall), specificity (selectivity), precision, accuracy and other metrics are computed for all samples, training sets, and test sets. In sample classification, Clasnip accepts inputs of genes, short fragments, contigs and even whole genomes. It can report classification probability and a multi-locus sequence typing table for SNPs. The classification performance was tested using short sequences of 16S, 16-23S and 50S rRNA regions for 12 haplotypes of Candidatus Liberibacter solanacearum (CLso), a regulated plant pathogen associated with severe disease in economically important Apiaceous and Solanaceous crops. The program was able to classify CLso samples with even only 1-2 SNPs available, and achieved 97.2%, 98.8% and 100.0% accuracy based on 16S, 16-23S, and 50S rRNA sequences, respectively. In comparison with all existing 12 haplotypes, we proposed that to be classified as a new haplotype, given samples have at least 2 SNPs in the combined region of 16S rRNA (OA2/Lsc2) and 16-23S IGS (Lp Frag 4-1611F/Lp Frag 4-480R) regions, and 2 SNPs in the 50S rplJ/rplL (CL514F/CL514R) regions. Besides, we have included the databases for differentiating Dickeya spp., Pectobacterium spp. and Clavibacter spp. In addition to bacteria, we also tested Clasnip performance on potato virus Y (PVY). 251 PVY genomes were 100% correctly classified into seven groups (PVYC, PVYN, PVYO, PVYNTN, PVYN:O, Poha, and Chile3). In conclusion, Clasnip is a statistically sound and user-friendly bioinformatic application for microorganism classification at the intraspecies level. Clasnip service is freely available at www.clasnip.com.

Efficient large-scale screening of viral pathogens by fragment length identification of pooled nucleic acid samples (FLIPNAS).

The necessity for the large-scale screening of viral pathogens has been amply demonstrated during the COVID-19 pandemic. During this time, SARS-CoV-2 nucleic acid pooled testing, such as Dorfman-based group testing, was widely adopted in response to the sudden increased demand for detection. However, the current approach still necessitates the individual retesting of positive pools. Here, we established an efficient method termed the fragment-length identification of pooled nucleic acid samples (FLIPNAS), where all subsamples (n = 8) can be uniquely labelled and tested in a single-time detection among pools of samples. We used a novel and simple design of unique primers (UPs) to generate amplicons of unique lengths after reverse transcription and polymerase chain reaction to reach this aim. As a result, the unique lengths of the amplicons can be recognized and traced back to the corresponding UPs and specific samples. Our results demonstrated that FLIPNAS could recognize one to eight positive subsamples in a single test without retesting positive pools. The system also showed sufficient sensitivity for the mass monitoring of SARS-CoV-2 and no cross-reactivity against three common respiratory diseases. Moreover, the FLIPNAS results of 40 samples with a positive ratio of 7.8% were in 100% agreement with their individual detection results using the gold standard. Collectively, this study shows that the efficiency of nucleic acid pooling detection can be further improved by FLIPNAS, which can speed up testing and mitigate the urgent demand for resources.

Isolation, Screening and Characterization of the Viral Pathogens from Apis mellifera Colonies

Isolation, Screening and Characterization of the Viral Pathogens from Apis mellifera Colonies

Screening of the Bacterial Pathogens in Biofloc Technology based Aquaculture of the Ctenopharyngodon idella

Great economic losses in fish aquaculture occur under the unhygienic conditions of the fishponds due to bacterial pathogens. Currently, Biofloc Technology (BFT) has proved successful in wastewater management as well as in controlling pathogenic loads. Since this technology has greatly supported marine fish, very scarce information is available for its successful implementation in freshwater fisheries. Furthermore, the pathogens specific to the carp cultures under the BFT system have not been studied yet. The unique attempt has been carried out in the Microbiology Lab of the Zoology Department of GC Women University, Faisalabad, Pakistan to screen bacterial pathogens in grass carp Ctenopharyngodon idella culture based on BFT utilizing agro-industrial wastes as a carbon source. The study confirmed the presence of bacterial isolates belonging to three genera namely Bacillus, Klebsiella, and Staphylococcus in water samples from three treatment groups. Bacillus species dominated over the pathogenic species i.e., Klebsiella and Staphylococcus in all treatment groups and is speculated to inhibit the harmful effects of Klebsiella and Staphylococcus species on the carp fish. This study is very important for the future designing of BFT based culture for freshwater fishes.

Evaluation of resazurin microplate method for rapid detection of vancomycin and linezolid resistance in Enterococcus faecalis and Enterococcus faecium clinical isolates.

Vancomycin and linezolid resistance among enterococci is an increasing problem due to a lack of alternative antibiotics. Early identification of vancomycin-resistant and linezolid-resistant strains can help prevent the spread of resistance to these antibiotics. Hence, early, rapid and accurate detection of vancomycin and linezolid resistance is critical. The resazurin microplate method (RMM) was developed for detecting vancomycin and linezolid susceptibility among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) clinical isolates, and its performance was further evaluated. A total of 209 non-duplicate clinical isolates and three strains from the faeces of domestic animals, including 142 E. faecalis (71 linezolid non-susceptible and 71 linezolid susceptible) and 70 E. faecium (23 vancomycin non-susceptible, 23 vancomycin susceptible, 12 linezolid non-susceptible and 12 linezolid susceptible), were tested using RMM. The susceptibility of E. faecium to vancomycin was detected within 5 h, with high susceptibility (23/23) and specificity (23/23). The susceptibility of E. faecalis and E. faecium to linezolid was detected within 4 h, with specificities of 98.59% and 100% and susceptibilities of 94.37% and 58.33% for E. faecalis and E. faecium, respectively. RMM had a good positive predictive value for the detection of vancomycin-non-susceptible E. faecium and linezolid-non-susceptible E. faecalis. It thus has the potential to become an alternative method for the rapid screening of these resistant pathogens in clinical practice.

A three-in-one hybrid nanozyme for sensitive colorimetric biosensing of pathogens

Pathogen screening is an important step in preventing foodborne diseases. In this study, a hybrid nanozyme, metal organic framework decorated with palladium (Pd) and platinum (Pt) (MIL-88@Pd/Pt), was innovatively synthesized and used with immune magnetic nanobeads (MNBs) for sensitive biosensing of Salmonella. First, immune MIL-88@Pd/Pt nanozymes and immune MNBs were mixed with target pathogens in a large-volume sample, resulting in effective isolation and specific label of target pathogens to form nanobead-Salmonella-nanozyme conjugates. Then, these conjugates were used to catalyze H2O2-TMB, and its color was changed from colorless to blue. Finally, catalysate absorption was measured to determine pathogen concentration. This colorimetric immunoassay could determine Salmonella typhimurium from 4 × 101 to 4 × 105 CFU/mL in 60 min with a detection limit of 32 CFU/mL.

Magnetorheological elastomer and smartphone enable microfluidic biosensing of foodborne pathogen

Rapid detection of foodborne pathogens is crucial to prevent the outbreaks of foodborne diseases. In this work, we proposed a novel microfluidic biosensor based on magnetorheological elastomer (MRE) and smartphone. First, micropump and microvalves were constructed by deforming the MRE under magnetic actuation and integrated into the microfluidic biosensor for fluidic control. Then, the micropump was used to deliver immune porous gold@platinum nanocatalysts (Au@PtNCs), bacterial sample, and immunomagnetic nanoparticles (MNPs) into a micromixer, where they were mixed, incubated and magnetically separated to obtain the Au@PtNC-bacteria-MNP complexes. After 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide were injected and catalyzed by the Au@PtNCs, smartphone was used to measure the color of the catalysate for quantitative analysis of target bacteria. Under optimal conditions, this biosensor could detect Salmonella typhimurium quantitatively and automatically in 1 h with a linear detection range of 8.0 × 101 CFU/mL to 8.0 × 104 CFU/mL and a detection limit of 62 CFU/mL. The microfluidic biosensor was compact in size, simple to use, and efficient for detection, and might be used for in-field screening of foodborne pathogens to prevent food poisoning.

Pathogen genomics study of an early medieval community in Germany reveals extensive co-infections

BackgroundThe pathogen landscape in the Early European Middle Ages remains largely unexplored. Here, we perform a systematic pathogen screening of the rural community Lauchheim “Mittelhofen,” in present-day Germany, dated to the Merovingian period, between fifth and eighth century CE. Skeletal remains of individuals were subjected to an ancient DNA metagenomic analysis. Genomes of the detected pathogens were reconstructed and analyzed phylogenetically.ResultsOver 30% of the individuals exhibit molecular signs of infection with hepatitis B virus (HBV), parvovirus B19, variola virus (VARV), and Mycobacterium leprae. Seven double and one triple infection were detected. We reconstructed four HBV genomes and one genome each of B19, VARV, and M. leprae. All HBV genomes are of genotype D4 which is rare in Europe today. The VARV strain exhibits a unique pattern of gene loss indicating that viruses with different gene compositions were circulating in the Early Middle Ages. The M. leprae strain clustered in branch 3 together with the oldest to-date genome from the UK.ConclusionsThe high burden of infectious disease, together with osteological markers of physiological stress, reflect a poor health status of the community. This could have been an indirect result of the climate decline in Europe at the time, caused by the Late Antique Little Ice Age (LALIA). Our findings suggest that LALIA may have created an ecological context in which persistent outbreaks set the stage for major epidemics of severe diseases such as leprosy and smallpox hundreds of years later.

Investigation of respiratory disease outbreaks of poultry in Bangladesh using two real-time PCR-based simultaneous detection assays.

For rapid and sensitive pathogen screening from field outbreaks, molecular techniques such as qPCR-based simultaneous detections are efficient. Respiratory diseases are the most detrimental diseases to the poultry industry and need to be addressed because of their major economic losses. In the current study, we have applied two different detection assays: one for simultaneous detection of avian influenza virus (AIV; M gene) and subtyping (H5, N1, H9, N2) using TaqMan probe chemistry (TaqMan multitarget) and another for simultaneous detection of Newcastle disease virus (NDV), infectious bronchitis virus (IBV), and infectious laryngotracheitis virus (ILTV) using SYBR Green chemistry (SYBR Green multitarget). Two individual qPCRs were conducted for the detection of four pathogens. Surveillance of tissue (n = 158) and oropharyngeal swab (206) samples from multiple poultry flocks during the years April 2020-July 2022 applying the TaqMan and SYBR Green multitarget qPCRs revealed that 48.9% of samples were positive for respiratory infections, of which 17.2% were positive for NDV, 25.5% were positive for AIV, 9.9% were positive for IBV, and only a single positive (0.3%) for ILTV. Among the AIV, 35% were highly pathogenic subtype H5N1 and 65% were low pathogenic subtype H9N2. Co-infections of 2-3 respiratory viruses were also accurately detected. Respiratory viral pathogens are quite common in Bangladeshi poultry and can be successfully detected using multitarget simultaneous real-time quantitative polymerase chain reaction (RT-qPCR) assays like those adopted in the current study. Increased mass surveillance, along with the molecular characterization of the circulating respiratory viruses, is crucial to control the epidemic and subsequently save the Bangladeshi poultry industry.

A Phi29-based unbiased exponential amplification and genotyping approach improves pathogen detection in tick samples.

Ticks are vectors for many infectious diseases, such as spotted fever group (SFG) rickettsioses and borreliosis, and are valuable in the study of pathogen ecology. Ticks have several growth stages that vary considerably in size; therefore, in most cases, DNA extracted from ticks is insufficient for subsequent studies, particularly for multiple pathogen screening and genotyping. Unbiased amplification of DNA from tick samples before analysis is a major requirement for subsequent ecological surveys and other studies. Phi29 DNA polymerase, an enzyme that exhibits strand displacement activity, can exponentially amplify DNA randomly, generating large quantities of DNA. In the present study, we developed a Phi29-based unbiased exponential amplification (PEA) assay to obtain sufficient tick DNA for genetic analysis. By using tick-borne pathogen detection and genotyping as a model, we tested and evaluated the feasibility of the assay. DNA was extracted from single ticks and subjected to PEA. The results showed that tick DNA could be amplified up to 105 fold. The amplified products were successfully used for pathogen screening and genotyping. Rickettsia was successfully detected and genotyped in samples with amplified DNA from single ticks. Furthermore, we identified a new genotype of Rickettsia from ticks collected from Dandong city, Liaoning province, Northeast China. This PEA assay is universal and can be extended to other applications where the quantity of DNA is greatly limited.

Identification, biological characterization, and fungicide screening of pathogens causing leaf spot of Belamcanda chinensis

The leaf spot of Belamcanda chinensis often appears in May to June and spreads rapidly during the flowering stage(July to September) in the cultivation fields, seriously affecting the yield and quality of B. chinensis. To identify and characterize the pathogens of the leaf spot, we isolated two species of Alternaria, identified them according to Koch's postulates, and tested their pathogenicity and biological characteristics. Furthermore, we determined the inhibitory effects of 6 chemical fungicides, 1 plant fungicide, and 3 microbial fungicides on the pathogens by using mycelial growth rate and plate confrontation method to select the appropriate control agents. The results showed that the two pathogens causing B. chinensis leaf spot were Alternaria tenuissima and A. alternata. The conidia of A. tenuissima often formed long chains with no or a few branches, while those of A. alternata often formed short branched chains. The optimum growth temperature of both A. tenuissima and A. alternata was 25 ℃. The two pathogens grew well in alkaline environment. The indoor fungicide screening experiments showed that 40% flusilazole had good inhibitory effects on the two pathogens, with the EC_(50) values of 12.42 mg·L~(-1) and 12.78 mg·L~(-1) for A. tenuissima and A. alternata, respectively. The results of this study provide a theoretical basis for the subsequent theoretical research and field control of B. chinensis leaf spot.

A Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Direct-from-Urine-Specimen Diagnostic for Gram-Negative Pathogens.

Urinary tract infections (UTIs) pose a major public health burden. The vast majority of UTIs are caused by Gram-negative bacteria. Current culture-based pathogen identification methods may require up to 24 to 48 h of incubation. In this study, we developed and evaluated a method for Gram-negative pathogen identification direct from urine, without culture, via matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in approximately 1 h. Urine samples were collected (n = 137) from the University of Maryland Medical Center clinical microbiology laboratory. To identify bacteria direct from urine, two methods were evaluated. First, 1 μL of urine was directly spotted onto the MALDI target plate, and second, 1 mL of urine was centrifuged at 8,000 rpm for 5 min before processing using the fast lipid analysis technique (FLAT). Mass spectra were acquired on the Bruker MALDI Biotyper sirius system in the negative-ion mode. Results were compared to those of standard culture methods. When 1 μL of urine was directly spotted, positive agreement was 81.5% (101/124) and, after centrifugation, 94.4% (117/124) relative to that of standard culture methods. Negative agreement for both methods was 100% (13/13). The time to results for both of the specimen preparation methods using the FLAT extraction protocol was approximately 1 h, with minimal hands-on time required (<5 min). The ability to rapidly identify pathogens directly from urine, without the need for culture, allows for faster turnaround times and, potentially, improved patient outcomes. Overall, the FLAT extraction protocol, in combination with lipid A identification, provides a reproducible and accurate method to rapidly identify urinary pathogens. IMPORTANCE This study describes and evaluates a direct-from-urine extraction method that allows identification of Gram-negative bacteria via MALDI-TOF MS within 1 h. Currently, identification of urinary pathogens requires 24 h of culture prior to identification. While this method may not replace culture, we demonstrate its utility in screening for common urinary pathogens. By providing identifications in under 1 h, clinicians can potentially treat patients sooner with more-targeted antimicrobial therapy. In turn, earlier treatment can improve patient outcome and antimicrobial stewardship. Furthermore, MADLI-TOF MS is a readily available, easy-to-use diagnostic tool in clinical laboratories, making implementation of this method possible.

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection.

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and β within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Staggered magnetic bead chains enhanced bacterial colorimetric biosensing

Pathogen screening is an important measure for ensuring food safety. A colorimetric biosensor was presented for Salmonella determination employing staggered magnetic bead chains for continuous-flow bacteria isolation, MnO2 nanozymes for effective signal amplification, and smartphone for accurate catalysate analysis. First, immune magnetic beads were distributed under the staggered magnetic field to form higher magnetic bead chains to occupy more cross-section of separation channel. Then, target bacteria were injected and captured onto magnetic bead chains, followed by labeling with the immune MnO2 nanozymes to form bead-bacteria-nanozyme complexes. Finally, H2O2-TMB substrate was catalyzed by MnO2 nanozymes on these complexes, and color change was monitored and analyzed through smartphone to quantify target bacteria. The sensor enabled Salmonella detection as few as 60 CFU/mL in 2 h.

Duplex detection of foodborne pathogens using a SERS optofluidic sensor coupled with immunoassay

Herein, we developed a surface-enhanced Raman spectroscopy (SERS) optofluidic sensor coupled with immunoprobes to simultaneously separate and detect the foodborne pathogens, Escherichia coli O157:H7, and Salmonella in lettuce and packed salad. The method consists of three steps of (i) enrichment to enhance detection sensitivity, (ii) selective separation and labelling of target bacteria by their specific antibody-bearing SERS-nanotags and (iii) detection of tagged bacterial cells using SERS within a hydrodynamic flow-focusing SERS optofluidic device, where even low counts of bacterial cells were detectable in the very thin-film-like sample stream. SERS-nanotags consisted of different Raman reporter molecules, representing each species, i.e., the detection of Raman reporter confirms the presence of the target pathogen. The anti-E. coli antibody used in this study functions against all strains of E. coli O157:H7 and the anti-Salmonella antibody used in this work acts on a wide range of Salmonella enterica strains. Bacterial counts of 1000, 100, and 10 CFU/ 200 g sample were successfully detected after only 15 min enrichment. Our method showed a very low detection limit value of 10 CFU/ 200 g sample for the bacterial mixture in both lettuce and packed salad, proving the efficiency and high sensitivity of our method to detect multiple pathogens in the food samples. The total analysis time, including sample preparation for simultaneous detection of multiple bacteria, was estimated to be 2 h, which is much less than the time required in conventional methods. Hence, our proposed protocol is considered a promising rapid and efficient approach for pathogen screening of food samples.

Unbiased screen for pathogens in human paraffin-embedded tissue samples by whole genome sequencing and metagenomics.

Identification of bacterial pathogens in formalin fixed, paraffin embedded (FFPE) tissue samples is limited to targeted and resource-intensive methods such as sequential PCR analyses. To enable unbiased screening for pathogens in FFPE tissue samples, we established a whole genome sequencing (WGS) method that combines shotgun sequencing and metagenomics for taxonomic identification of bacterial pathogens after subtraction of human genomic reads. To validate the assay, we analyzed more than 100 samples of known composition as well as FFPE lung autopsy tissues with and without histological signs of infections. Metagenomics analysis confirmed the pathogenic species that were previously identified by species-specific PCR in 62% of samples, showing that metagenomics is less sensitive than species-specific PCR. On the other hand, metagenomics analysis identified pathogens in samples, which had been tested negative for multiple common microorganisms and showed histological signs of infection. This highlights the ability of this assay to screen for unknown pathogens and detect multi-microbial infections which is not possible by histomorphology and species-specific PCR alone.

Visual closed dumbbell-mediated isothermal amplification (CDA) for on-site detection of Rickettsia raoultii.

Spotted fever group (SFG) rickettsioses are important zoonoses, threatening human health seriously and gradually attracting more attention in the world. SFG rickettsiae are classified as neglected pathogens. If these pathogens are detected at all, they are usually recognized very late in the infection through indirect detection of specific antibodies. Previous studies have shown that Rickettsia raoultii (R. raoultii), a member of the SFG rickettsiae, occurs with increasing incidence in remote countries. Therefore, a rapid detection method for R. raoultii is in urgently need. In this study, a R. raoultii diagnosis method by closed dumbbell-mediated isothermal amplification (R-CDA) assay targeting a conserved sequence of the outer membrane protein A (OmpA) gene with high sensitivity and specificity was developed. This assay offered a rapid and simple method for on-site detection of R. raoultii. Firstly, four pairs of R-CDA primers were designed and the optimum primer set was selected to amplify target gene specifically and effectively. Then, a pair of outer primer was designed to accelerate the reaction based on the inner primers to establish the RO-CDA reaction. In addition, the results of real-time amplification curves, melting curves and end-point colorimetric judgements showed that the established visual RO-CDA reaction could accurately detect R. raoultii without cross-reaction with other closely related pathogens. Furthermore, the detection limit of visual RO-CDA assay was 10 copies/μL, which was feasible for on-site detection with merits of easy-operation, rapidity, high sensitivity, and specificity. In conclusion, the developed RO-CDA detection method could be helpful for pathogen screening and epidemic prevention at the point of care.