Methods For Detection Of Pathogens Research Articles

Development of detection methods for soil borne pathogens

Development of detection methods for soil borne pathogens

Utilizing metagenomic next-generation sequencing for diagnosis and lung microbiome probing of pediatric pneumonia through bronchoalveolar lavage fluid in pediatric intensive care unit: results from a large real-world cohort.

Metagenomic next-generation sequencing (mNGS) is a powerful method for pathogen detection in various infections. In this study, we assessed the value of mNGS in the pathogen diagnosis and microbiome analysis of pneumonia in pediatric intensive care units (PICU) using bronchoalveolar lavage fluid (BALF) samples. A total of 104 pediatric patients with pneumonia who were admitted into PICU between June 2018 and February 2020 were retrospectively enrolled. Among them, 101 subjects who had intact clinical information were subject to parallel comparison of mNGS and conventional microbiological tests (CMTs) for pathogen detection. The performance was also evaluated and compared between BALF-mNGS and BALF-culture methods. Moreover, the diversity and structure of all 104 patients' lung BALF microbiomes were explored using the mNGS data. Combining the findings of mNGS and CMTs, 94.06% (95/101) pneumonia cases showed evidence of causative pathogenic infections, including 79.21% (80/101) mixed and 14.85% (15/101) single infections. Regarding the pathogenesis of pneumonia in the PICU, the fungal detection rates were significantly higher in patients with immunodeficiency (55.56% vs. 25.30%, P =0.025) and comorbidities (40.30% vs. 11.76%, P=0.007). There were no significant differences in the α-diversity either between patients with CAP and HAP or between patients with and without immunodeficiency. Regarding the diagnostic performance, the detection rate of DNA-based BALF-mNGS was slightly higher than that of the BALF-culture although statistically insignificant (81.82% vs.77.92%, P=0.677) and was comparable to CMTs (81.82% vs. 89.61%, P=0.211). The overall sensitivity of DNA-based mNGS was 85.14% (95% confidence interval [CI]: 74.96%-92.34%). The detection rate of RNA-based BALF-mNGS was the same with CMTs (80.00% vs 80.00%, P>0.999) and higher than BALF-culture (80.00% vs 52.00%, P=0.045), with a sensitivity of 90.91% (95%CI: 70.84%-98.88%). mNGS is valuable in the etiological diagnosis of pneumonia, especially in fungal infections, and can reveal pulmonary microecological characteristics. For pneumonia patients in PICU, the mNGS should be implemented early and complementary to CMTs.

Rationalize the choose of pathogen detection methods to conduct precise anti-infection therapy

Rationalize the choose of pathogen detection methods to conduct precise anti-infection therapy

The Utility of Metagenomic Next-Generation Sequencing (mNGS) in the Management of Patients With Bronchiectasis: A Single-Center Retrospective Study of 93 Cases.

Bronchiectasis is a chronic inflammatory respiratory disease mainly caused by pathogenic infections. However, standard methods of pathogen detection show prolonged cycle durations and unsatisfactory sensitivity and detection rates. Macrogenomic next-generation sequencing (mNGS) emerges as a promising technique for swift, effective, and unbiased pathogen detection and subsequent data interpretation. Here, a retrospective analysis of 93 patients with suspected bronchiectasis was performed to assess the clinical applicability of mNGS. Bronchoalveolar alveolar lavage fluid (BALF) samples were collected from these subjects, followed by standard assays and mNGS separately. The turnaround time, detection rate, and pathogen identification using mNGS were compared with those of standard methods. mNGS identified a greater number of bacteria (72 vs 16), fungi (26 vs 19), and viruses (14 vs 0) than standard methods. Specifically, the commonly identified bacteria were Haemophilus, Mycobacterium intracellulare, Pseudomonas, and Streptococcus pneumoniae, while the most detected fungi were Aspergillus and the most prevalent viruses were human herpesviruses. Of note, 29 out of 30 patients (96.67%) who received optimized treatment strategies based on mNGS results experienced recovery. Collectively, these findings suggest that mNGS has the potential to improve the diagnosis and treatment of bronchiectasis patients by enabling rapid and precise pathogen detection, which can lead to timely and effective treatment strategies.

Novel next generation sequencing panel method for the multiple detection and identification of foodborne pathogens in agricultural wastewater.

Detecting and identifying the origins of foodborne pathogen outbreaks is a challenging. The Next-Generation Sequencing (NGS) panel method offers a potential solution by enabling efficient screening and identification of various bacteria in one reaction. In this study, new NGS panel primer sets that target 18 specific virulence factor genes from six target pathogens (Bacillus cereus, Yersinia enterocolitica, Staphylococcus aureus, Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus) were developed and optimized. The primer sets were validated for specificity and selectivity through singleplex PCR, confirming the expected amplicon size. Crosscheck and multiplex PCR showed no interference in the primer set or pathogenic DNA mixture. The NGS panel analysis of spiked water samples detected all 18 target genes in a single reaction, with pathogen concentrations ranging from 108 to 105 colony-forming units (CFUs) per target pathogen. Notably, the total sequence read counts from the virulence factor genes showed a positive association with the CFUs per target pathogen. However, the method exhibited relatively low sensitivity and occasional false positive results at low pathogen concentrations of 105 CFUs. To validate the detection and identification results, two sets of quantitative real-time PCR (qPCR) analyses were independently performed on the same spiked water samples, yielding almost the same efficiency and specificity compared to the NGS panel analysis. Comparative statistical analysis and Spearman correlation analysis further supported the similarity of the results by showing a negative association between the NGS panel sequence read counts and qPCR cycle threshold (Ct) values. To enhance NGS panel analysis for better detection, optimization of primer sets and real-time NGS sequencing technology are essential. Nonetheless, this study provides valuable insights into applying NGS panel analysis for multiple foodborne pathogen detection, emphasizing its potential in ensuring food safety.

GIS-BASED INTELLIGENT DECISION-MAKING SUPPORT SYSTEM FOR THE DISASTER RESPONSE OF INFECTIOUS DISEASES

Abstract. The study aims to develop GIS technology for managing pathogen contamination disaster situations to cope with the occurrence of disaster situations caused by the climate crisis. This study was conducted in a situation where the budgets of institutions with many emergency rescue workers are being invested worldwide, and there are various risks such as water pollution due to climate change, so it is necessary to develop technologies to respond to them.In this study, methods and technical means for emergency response to pathogen contamination were prepared through professional analysis results based on spatial information, and rapid and accurate pathogen detection, situation recognition, event control and response methods, risk assessment and epidemiological investigation methods, and technologies were developed.Technological means include collecting and analyzing water quality data using satellites and drones, and evaluating the severity and degree of water pollution using social media data and photos. Through these technologies, a GIS platform that can effectively manage and provide data sources, data collection, interoperability, data harmonization, data management, and data processing is established, and commercialization is aimed at real users and field-oriented. These technology developments enable rapid response and response in disaster situations, and can reduce threats to life and safety.

Combination of transbronchial cryobiopsy based clinic-radiologic-pathologic strategy and metagenomic next-generation sequencing for differential diagnosis of rapidly progressive diffuse parenchymal lung diseases.

The complicated spectrum of rapidly progressive diffused parenchymal lung diseases (RP-DPLD) creates obstacles to the precise diagnosis and treatment. We evaluated the differential diagnostic value of transbronchial cryobiopsy (TBCB) based clinic-radiologic-pathologic (CRP) strategy combined with bronchoalveolar lavage fluid (BALF) metagenomic next-generation sequencing (mNGS) in RP-DPLD patients. RP-DPLD patients who underwent the diagnostic strategy of TBCB-based CRP combined with BALF mNGS at Shanghai East Hospital from May 2020 to Oct 2022 were retrospectively analyzed. Clinical characteristics were summarized, including demographic data, high-resolution computed tomography (HRCT) findings, histopathology of TBCB and microbiological results. Diagnostic value of the combined strategy, as well as the sensitivity, specificity, and positive detection rates of mNGS were evaluated. A total of 115 RP-DPLD patients were enrolled, with a mean age of 64.4 years old and a male proportion of 54.8%. The pulmonary imaging findings in most patients were complex and diverse, with all patients showing bilateral lung diffuse lesions in HRCT, and progressively aggravated imaging changes within one month. After combining TBCB-based CRP strategy with mNGS, all participants received a corresponding diagnosis with 100% diagnostic yield. In these patients, 58.3% (67/115) were diagnosed with noninfectious RP-DPLD and 41.7% (48/115) with infection-related RP-DPLD. There were 86.1% of cases with known etiology according to the DPLD classification. BALF mNGS and traditional pathogen detection methods were performed in all patients, the positive detection rates were 50.4% (58/115) and 32.2% (37/115), respectively. Meanwhile, the mNGS showed significantly higher sensitivity and negative predictive value than the traditional pathogen detection methods for the diagnosis of infection-related RP-DPLD (100% vs 60.4% (p<0.001), 100% vs 75.6% (p<0.001), respectively). Among noninfectious RP-DPLD patients, the true negative rate of mNGS was 85.1% (57/67). All patients had their treatment regimen modified and the 30-day mortality was 7.0%. The novel strategy of TBCB-based CRP combined with mNGS provided dependable and sufficient evidence for the diagnosis, meanwhile further improved the accuracy of RP-DPLD treatment, as well as the prognosis of patients. Our results highlight the significant value of combined strategy in determining whether the RP-DPLD patients were infection associated or not.

User-friendly, signal-enhanced planar spiral coil-based magnetoelastic biosensor combined with humidity-resistant phages for simultaneous detection of Salmonella Typhimurium and Escherichia coli O157:H7 on fresh produce

A user-friendly, signal-enhanced magnetoelastic (ME) biosensor was newly constructed, consisting of three sensors, a movable pencil-type planar spiral coil, and a set of hand-held signal amplifiers. It was combined with humidity-resistant, wild-type phages instead of humidity-susceptible E2 phage to simultaneously detect Salmonella Typhimurium (ST) and Escherichia coli O157:H7 (EC) on the surface of an apple. The set of hand-held signal amplifiers, four times smaller than those in the planar spiral coil-based ME biosensor, was constructed with an optimal DC magnetic field strength of 76 G. These optimized hand-held signal amplifiers enhanced the signal amplitude of the sensor by up to 1.5 times, enabling the distinction of individual peaks from three sensors. Both phage-immobilized sensors detected each target specifically under ambient humidity without the need for a bulky humidifier. The optimized exposure time for phage binding with each target on fresh produce was 16 min, almost twice as fast as the rectangular solenoid coil-based ME biosensor. The signal-enhanced ME biosensor detected its target bacteria from a mixture of ST and EC inoculated on an apple simultaneously with an enhanced detection limit of 1.7 ± 0.4 log CFU/25 mm2 for ST and 1.6 ± 0.3 log CFU/25 mm2 for EC. Notably, the detection limit and sensitivity did not show significant differences between each single analyte and bacterial mixture, indicating no interference with another coexisting pathogens. Our findings highlight the signal-enhanced ME biosensor as a rapid, efficient, specific, and user-friendly method for the simultaneous detection of fresh produce-borne pathogens.

Fully integrated sample-in-answer-out platform for viral detection using digital reverse transcription recombinase polymerase amplification (dRT-RPA)

Recombinase polymerase amplification (RPA) is one of the most promising diagnostic methods for pathogen detection, owing to the simplified isothermal amplification technique. Using one-step digital reverse transcription RPA (dRT-RPA) to detect viral RNA provides a fast diagnosis and absolute quantification. Here, we present a chip that purifies, digitalizes, and detects viral RNA of SARS-CoV-2 in a fully automated and sensitive manner. The chip purifies the RNA using the surface charge concept of magnet bead-RNA binding, then mixes the RNA with the amplification reagents, digitalizes the amplification mixture, and performs dRT-RPA. RNA-bead complex is transported among purification buffers that are separated by an oil phase. For reagent manipulation and mixing, a magnetic valve system is integrated on the chip, where an external magnet controls the reagent direction and time of addition. Besides, a novel vacuum system is suggested to drive and regulate the reagents into two fluid systems simultaneously in ∼2 min. We also developed a cost-effective way to perform fluorescent detection for dRT-RPA on chip by using EvaGreen® dye. With integrated heating and optical detection system, the on-chip dRT-RPA presents a sample-to-answer detection platform for absolute viral RNA quantitation in 37 min and a sensitivity as low as 10 RNA copies/μL. Hence, this platform is expected to be a useful tool for accurate and automated diagnosis of infectious diseases.

MXene@Au based electrochemical biosensor with pretreatment by magnetic nanoparticles for determination of MRSA from clinical samples

Pathogenic bacteria are associated with high morbidity rates and present significant diagnostic challenges in terms of rapid detection. This study introduces a magnetic separation-based electrochemical biosensor for the detection of Methicillin-resistant Staphylococcus aureus (MRSA). Vancomycin (Van) was used to modify on the surface of polyethyleneimine (PEI) mediated MBs (MBs-PEI-Van) for separation and enrichment of MRSA. The MBs-PEI-Van shown a satisfactory stability and applicability with capture effective (CE) > 85% in both PBS and cerebrospinal fluid (CSF) samples. MXene@Au with controllable size of AuNPs was synthesized by a self-reduction method and employed to modify the glassy carbon electrode (GCE). Immunoglobulin G (IgG) was loaded onto the modified electrode to immobilize MRSA, and ferroceneboronic acid (Fc-BA) was used as a probe for quantitative determination. The differential pulse voltammetry (DPV) current was plotted against the concentration of MRSA from 3.8 × 101 to 3.8 × 107 CFU/mL with a limit of detection (LOD) of 3.8 × 101 CFU/mL. In addition, MRSA was successfully detected in spiked CSF samples with satisfactory recoveries (94.35–107.81 %) and validation results (RSD < 11 %). Overall, this study presents a promising method for the detection of MRSA, with the potential to be further developed into a universal pathogen detection method.

Nondestructive Methods of Pathogen Detection: Importance of Mosquito Integrity in Studies of Disease Transmission and Control.

Mosquitoes are vectors of many pathogens, including viruses, protozoans, and helminths, spreading these pathogens to humans as well as to wild and domestic animals. As the identification of species and the biological characterization of mosquito vectors are cornerstones for understanding patterns of disease transmission, and the design of control strategies, we conducted a literature review on the current use of noninvasive and nondestructive techniques for pathogen detection in mosquitoes, highlighting the importance of their taxonomic status and systematics, and some gaps in the knowledge of their vectorial capacity. Here, we summarized the alternative techniques for pathogen detection in mosquitoes based on both laboratory and field studies. Parasite infection and dissemination by mosquitoes can also be obtained via analyses of saliva- and excreta-based techniques or of the whole mosquito body, using a near-infrared spectrometry (NIRS) approach. Further research should be encouraged to seek strategies for detecting target pathogens while preserving mosquito morphology, especially in biodiversity hotspot regions, thus enabling the discovery of cryptic or new species, and the determination of more accurate taxonomic, parasitological, and epidemiological patterns.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

Triplex qPCR assay for Campylobacter jejuni and Campylobacter coli monitoring in wastewater

Campylobacter spp. is one of the most frequent pathogens of bacterial gastroenteritis recorded worldwide. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the two major disease-associated species, accounting for >95 % of infections, and thus have been selected for disease surveillance. Monitoring temporal variations in pathogen concentration and diversity excreted from community wastewater allows the early detection of outbreaks. Multiplex real-time/quantitative PCR (qPCR) enables multi-target quantification of pathogens in various types of samples including wastewater. Also, an internal amplification control (IAC) is required for each sample when adopting PCR-based methods for pathogen detection and quantification in wastewater to exclude the inhibition of the wastewater matrix. To achieve reliable quantification of C. jejuni and C. coli towards wastewater samples, this study developed and optimized a triplex qPCR assay by combining three qPCR primer-probe sets targeting Campylobacter jejuni subsp. jejuni, Campylobacter coli, and Campylobacter sputorum biovar sputorum (C. sputorum), respectively. This triplex qPCR assay not only can directly and simultaneously detect the concentration of C. jejuni and C. coli in wastewater but also can achieve the PCR inhibition control using C. sputorum primer-probe set. This is the first developed triplex qPCR assay with IAC for C. jejuni and C. coli, to be used in the wastewater-based epidemiology (WBE) applications. The optimized triplex qPCR assay enables the detection limit of the assay (ALOD100%) and wastewater (PLOD80%) as 10 gene copy/μL and 2 log10 cells/mL (2 gene copies/μL of extracted DNA), respectively. The application of this triplex qPCR to 52 real raw wastewater samples from 13 wastewater treatment plants demonstrated its potential as a high-throughput and economically viable tool for the long-term monitoring of C. jejuni and C. coli prevalence in communities and the surrounding environments. This study provided an accessible methodology and a solid foundation for WBE-based monitoring of Campylobacter spp. relevant diseases and paved the road for future WBE back-estimation of C. jejuni and C. coli prevalence.

Multiple mutations of Mycobacterium intracellulare subsp. chimaera causing false-negative reaction to the transcription-reverse transcription concerted method for pathogen detection

ObjectivesTo report an isolate of Mycobacterium intracellulare subsp. chimaera with multiple mutations in 16S ribosomal RNA (rRNA) gene, resulting in the false-negative reaction to the transcription-reverse transcription concerted (TRC) method for Mycobacterium avium-intracellulare complex. MethodsWe used TRC, polymerase chain reaction (PCR), and Matrix-assisted laser desorption/ionization Time-of-Flight/Mass Spectrometry (MALDI-TOF/MS) methods to identify a clinical isolate in 2021. Due to the discordant results between TRC and PCR or MALDI-TOF MS methods, 16S rRNA sequencing, whole-genome shotgun (WGS) sequencing, and average nucleotide identity (ANI) analysis were employed to identify the isolate. ResultsA mycobacterial isolate from a sputum sample gave negative results for the detection of Mycobacterium tuberculosis complex or M. avium-intracellulare complex by the TRC method. However, the isolate was identified as M. intracellulare by both PCR method and MALDI-TOF MS method. WGS sequencing of 16S rRNA genome revealed eight substitution mutations and one insertion mutation within the region, which could hamper the correct reaction to TRC method. Subsequent ANI analysis between the isolate and various species of nontuberculosis mycobacteria revealed that the isolate could be identified as M. intracellulare subsp. chimaera. ConclusionRare mutations within the 16S rRNA genome resulted in the false-negative identification of Mycobacterium chimaera by the TRC method. WGS sequencing and ANI analysis was necessary to identify the isolate.

A Review of Modern Methods for the Detection of Foodborne Pathogens.

Despite the recent advances in food preservation techniques and food safety, significant disease outbreaks linked to foodborne pathogens such as bacteria, fungi, and viruses still occur worldwide indicating that these pathogens still constitute significant risks to public health. Although extensive reviews of methods for foodborne pathogens detection exist, most are skewed towards bacteria despite the increasing relevance of other pathogens such as viruses. Therefore, this review of foodborne pathogen detection methods is holistic, focusing on pathogenic bacteria, fungi, and viruses. This review has shown that culture-based methods allied with new approaches are beneficial for the detection of foodborne pathogens. The current application of immunoassay methods, especially for bacterial and fungal toxins detection in foods, are reviewed. The use and benefits of nucleic acid-based PCR methods and next-generation sequencing-based methods for bacterial, fungal, and viral pathogens' detection and their toxins in foods are also reviewed. This review has, therefore, shown that different modern methods exist for the detection of current and emerging foodborne bacterial, fungal, and viral pathogens. It provides further evidence that the full utilization of these tools can lead to early detection and control of foodborne diseases, enhancing public health and reducing the frequency of disease outbreaks.

Inferring pathogen presence when sample misclassification and partial observation occur

Abstract Surveillance programmes are essential for detecting emerging pathogens and often rely on molecular methods to make inference about the presence of a target disease agent. However, molecular methods rarely detect target DNA perfectly. For example, molecular pathogen detection methods can result in misclassification (i.e. false positives and false negatives) or partial detection errors (i.e. detections with ‘ambiguous’, ‘uncertain’ or ‘equivocal’ results). Then, when data are to be analysed, these partial observations are either discarded or censored; this, however, disregards information that could be used to make inference about the true state of the system. There is a critical need for more direction and guidance related to how many samples are enough to declare a unit of interest ‘pathogen free’. Here, we develop a Bayesian hierarchal framework that accommodates false negative, false positive and uncertain detections to improve inference related to the occupancy of a pathogen. We apply our modelling framework to a case study of the fungal pathogen Pseudogymnoascus destructans (Pd) identified in Texas bats at the invasion front of white‐nose syndrome. To improve future surveillance programmes, we provide guidance on sample sizes required to be 95% certain a target organism is absent from a site. We found that the presence of uncertain detections increased the variability of resulting posterior probability distributions of pathogen occurrence, and that our estimates of required sample size were very sensitive to prior information about pathogen occupancy, pathogen prevalence and diagnostic test specificity. In the Pd case study, we found that the posterior probability of occupancy was very low in 2018, but occupancy probability approached 1 in 2020, reflecting increasing prior probabilities of occupancy and prevalence elicited from the site manager. Our modelling framework provides the user a posterior probability distribution of pathogen occurrence, which allows for subjective interpretation by the decision‐maker. To help readers apply and use the methods we developed, we provide an interactive RShiny app that generates target species occupancy estimation and sample size estimates to make these methods more accessible to the scientific community (https://rmummah.shinyapps.io/ambigDetect_sampleSize). This modelling framework and sample size guide may be useful for improving inferences from molecular surveillance data about emerging pathogens, non‐native invasive species and endangered species where misclassifications and ambiguous detections occur.

Metagenomic next-generation sequencing performed on blood samples for the early recognition of severe Pneumocystis pneumonia in critical hematological patients

Severe Pneumocystis pneumonia (PCP) has a poor prognosis, and its early and precise diagnosis is difficult in immunocompromised individuals. Therefore, this study explored the diagnostic value of metagenomic next-generation sequencing (mNGS) of peripheral blood in diagnosing severe PCP in patients with hematological diseases. This prospective study analyzed the clinical manifestations, mNGS results (from the peripheral blood), traditional pathogen detection results, laboratory test results, chest computed tomography (CT) images, treatments, and outcomes of severe PCP in hematological patients who were hospitalized in the 2 centers of the Affiliated Hospital of Soochow University between September 2019 and October 2021. A total of 31 cases of hematological diseases complicated with pulmonary infections, including 7 cases of severe PCP diagnosed by mNGS performed on peripheral blood samples, were analyzed. Traditional pathogen detection methods for PCP cannot be used. In contrast, the laboratory readings for Pneumocystis jirovecii (Pj) detected within 48 hours of symptom onset by mNGS on the 7 blood samples ranged from 12 to 5873, with a median value of 43. Under the guidance of the mNGS results, preemptive antimicrobial therapy with trimethoprim/sulfamethoxazole alone or in combination with caspofungin was administered to treat Pj. After treatment, 4 patients recovered, and 3 patients died of acute respiratory failure and acute respiratory distress syndrome (ARDS). MNGS performed on peripheral blood samples is optional but can provide early recognition of severe PCP and help guide empirical treatment in critical hematological patients.

Increased growth temperature and vitamin B12 supplementation reduces the lag time for rapid pathogen identification in BHI agar and blood cultures.

Background: Rapid diagnostics of pathogens is essential to prescribe appropriate antibiotic therapy. The current methods for pathogen detection require the bacteria to grow in a culture medium, which is time-consuming. This increases the mortality rate and global burden of antimicrobial resistance. Culture-free detection methods are still under development and are not common in the clinical routine. Therefore, decreasing the culture time for accurately detecting infection and resistance is vital for diagnosis. Methods: This study investigated easy-to-implement factors (in a minimal laboratory set-up), including inoculum size, incubation temperature, and additional supplementation ( e.g., vitamin B12 and trace metals), that can significantly reduce the bacterial lag time (t lag). These factors were arranged in simple two-level factorial designs using Gram-positive cocci ( Staphylococcus aureus), Gram-positive bacilli ( Bacillus subtilis), and Gram-negative bacilli ( Escherichia coli and Pseudomonas aeruginosa) bacteria, including clinical isolates with known antimicrobial resistance profiles. Blood samples spiked with a clinical isolate of E. coli CCUG 17620 (Culture Collection University of Gothenburg) were also tested to see the effect of elevated incubation temperature on bacterial growth in blood cultures. Results: We observed that increased incubation temperature (42°C) along with vitamin B12 supplementation significantly reduced the t lag (10 - 115 minutes or 4% - 49%) in pure clinical isolates and blood samples spiked with E. coli CCUG17620. In the case of the blood sample, PCR results also detected bacterial DNA after only 3h of incubation and at three times the CFU/mL. Conclusion: Enrichment of bacterial culture media with growth supplements such as vitamin B12 and increased incubation temperature can be a cheap and rapid method for the early detection of pathogens. This proof-of-concept study is restricted to a few bacterial strains and growth conditions. In the future, the effect of other growth conditions and difficult-to-culture bacteria should be explored to shorten the lag phase.

Sensitivity and Specificity of Molecular-Based Pathogen Detection Method in Refillable Drinking Water Samples

The existence of refillable drinking water depots helps the community to get affordable and practical drinking water. However, poor quality drinking water will, however, have an effect on health. One of the quality parameters of drinking water that is suitable for consumption is not contaminated by the bacteria Escherichia coli, Salmonella sp. and Escherichia sp. Measurement of the quality of drinking water, in addition to microbiological tests, can be carried out molecularly using PCR (Polymerase Chain Reaction) method. Therefore, the aim of this study was to examine the sensitivity and specificity of PCR for detection of drinking water pathogens. DNA was extracted from cultures of E. coli, Salmonella sp., Escherichia sp. and some non-coliform bacteria. PCR was performed separately using primer pairs of E. coli-AA-Forward and E.coli-AA-Reverse, Salmonella-OY-Forward and Salmonella-OY-Reverse, E. coli-DB-Forward and E. coli-DB-Reverse. The results of the PCR sensitivity showed that the minimum amount of DNA that can be detected by this method were 0.0025 ng/µL, 0,0005 ng/µL, 0,04 ng/µL for E. coli, Salmonella sp., Escherichia sp., respectively. The results of the PCR specificity of each primer pairs indicated that these methods were able to detect each coliform bacterium specifically according to PCR product size of ± 417 bp, ± 559 bp and ± 815 bp for E. coli, Salmonella sp., Escherichia sp., respectively.

Rapid and Sensitive Fluorescence Detection of Staphylococcus aureus Based on Polyethyleneimine-Enhanced Boronate Affinity Isolation.

There are increasing demands for fast and simple detection of pathogens in foodstuffs. Fluorescence analysis has demonstrated significant advantages for easy operation and high sensitivity, although it is usually hindered by a complex matrix, low bacterial abundance, and long-term bacterial enrichment. Effective enrichment procedures are required to meet the requirements for food detection. Here, boronate-functionalized cellulose filter paper and specific fluorescent probes were combined. An integrated approach for the enrichment of detection of Staphylococcus aureus was proposed. The modification of polyethyleneimine demonstrated a significant effect in enhancing the bacterial enrichment, and the boronate affinity efficiency of the paper was increased by about 51~132%. With optimized conditions, the adsorption efficiency for S. aureus was evaluated as 1.87 × 108 CFU/cm2, the linear range of the fluorescent analysis was 104 CFU/mL~108 CFU/mL (R2 = 0.9835), and the lowest limit of detection (LOD) was calculated as 2.24 × 102 CFU/mL. Such efficiency was validated with milk and yogurt samples. These results indicated that the material had a high enrichment capacity, simple operation, and high substrate tolerance, which had the promising potential to be the established method for the fast detection of food pathogens.