Method For Detection Of Bacteria Research Articles

A rapid and efficient DNA isolation method for qPCR-based detection of pathogenic and spoilage bacteria in milk

The objective of this study was to find an efficient, rapid, simple, and cost-effective method of pretreating raw milk samples to produce PCR-ready DNA for subsequent microbial detection using the strains of eight bacterial species. A total of 17 in-house protocols and three commercial kits were evaluated in three steps from scientific, practical, and economic perspectives. The results showed that an in-house procedure involving Triton X-100-based pretreatment and an inhibitor removal resin was superior to all other methods tested in terms of DNA yield, sensitivity, ease of sample handling, time efficiency, and cost per sample. Overall, this simplified preanalytical protocol was shown to have a great potential for use in rapid detection of dairy-related bacterial species, thereby enabling early intervention in the food chain and thus reducing the risk of negative economic and health outcomes. • Methods of pretreating raw milk samples to yield PCR-ready bacterial DNA were tested. • Of 20 procedures, a Triton X-100-based in-house protocol showed the best results. • This approach has a great potential for use in detection of dairy-related bacteria. • With further optimization, the protocol may be included in milk quality guidelines.

High-Throughput Flow-Through Direct Immunoassays for Targeted Bacteria Detection.

Regulatory authorities require analytical methods for bacteria detection to analyze large sample volumes (typically 100 mL). Currently only the Membrane Filtration and the Most Probable Number assays analyze such large volumes, while other assays for bacteria detection (ELISA, lateral flow assays, etc.) typically analyze volumes 1000 times smaller. This study describes flow-through direct immunoassays (FTDI), a new methodology for the targeted detection of bacteria in liquid samples of theoretically any volume. Flow-through direct immunoassays are performed in fluid-permeable microwells (e.g., wells of a filter well plate) that have a membrane on their bottom where the bacteria are trapped before their detection using a direct immunoassay. Two versions of FTDI assays for the detection of E. coli in 10 mL of sample were developed. A rapid FTDI assay that can be completed in less than 2.5 h can detect E. coli bacteria in levels down to 17 CFU/mL, and an ultrasensitive FTDI assay that employs an additional bacteria culturing step to boost the sensitivity can detect E. coli bacteria in levels lower than 1 CFU/mL in less than 5.5 h. All the steps of the assays, including the immunoassay steps, the culturing step, and the analytical signal measurement step are performed inside the well plate to decrease the chance of contamination and ensure a safe, easy process for the user. The assays were assessed and validated in tap water, river water, and apple juice samples, and the results suggests that the assays are robust, precise, and accurate. When the assays are performed in 96-well filter plates, a filter well plate vacuum manifold and a multichannel peristaltic pump are also used, so multiple samples can be analyzed in parallel to allow high-throughput analysis of samples.

The Sensitivity and Specificity of Loop-Mediated Isothermal Amplification and PCR Methods in Detection of Foodborne Microorganisms: A Systematic Review and Meta-Analysis.

Background:The loop-mediated isothermal amplification (LAMP) method is frequently used for identifying many microorganisms. The present review aimed to evaluate the sensitivity and specificity of LAMP method for detection of food-borne bacteria and to compare these features with those of polymerase chain reaction (PCR), as an alternative molecular diagnostic procedure, and with cultivation method, as the gold standard method.Methods:The literature was searched in electronic databases (PubMed, Scopus, Web of Science, and EMBASE) for recruiting publications within Jan 2000 to Jul 2021. We used the combinations of keywords including foodborne disease, LAMP, PCR, Loop-mediated isothermal amplification, and polymerase chain reaction. Meta-analysis was used to adjust the correlation and heterogeneity between the studies. The efficiency of the methods was presented by negative likelihood ratio, positive likelihood ratio, sensitivity, specificity, and odds ratio using forest plots. A P-value less than 0.05 was considered as statistical significance cut off. The confidence intervals were presented at the 95% interval.Results:Overall, 23 relevant studies were analyzed. The sensitivities of LAMP and PCR methods were estimated to be 96.6% (95% CI: 95.0–97.7) and 95.6% (95%CI: 91.5–97.8), respectively. The specificities of LAMP and PCR were also estimated to be 97.6% (95%CI: 92.6–99.3) and 98.7% (95%CI: 96.5–99.5), respectively.Conclusion:The specificities of LAMP and PCR assays were determined by comparing their results with cultivation method as the gold standard. Overall, the specificity of both PCR and LAMP methods was low for detection of fastidious bacteria. Nevertheless, LAMP and PCR methods have acceptable specificities and sensitivities, and their application in clinical practice necessitates more studies.

A platform for specific and sensitive detection of target bacteria by selective magnetic enrichment and a broad-spectrum fluorescent probe

The indiscriminate application of antibiotics has led to the spread of antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and Klebsiella pneumoniae carbapenemase 2-expressing Klebsiella pneumoniae (KPC-2 KP), resulting in a global threat to public health. A method for specific, sensitive, and rapid detection of such bacteria is essential for clinical diagnosis and treatment. In this study, target bacteria were selectively captured using aptamer-coated magnetic beads (Apt-MBs), followed by recognition and detection by a broad-spectrum fluorescent probe, fragment crystallizable mannose binding lectin-coated gold nanoclusters (FcMBL@AuNCs). As a proof of concept study, a Gram-positive bacterium (i.e., MRSA) and Gram-negative bacterium (i.e., KPC-2 KP) were selected as models and detected by the proposed strategy. The determination of MRSA and KPC-2 KP was in the range of 20–108 CFU/mL with a detection limit (DL) of ~20 CFU/mL. Furthermore, the method shows great specificity for target bacteria, and detection procedures could be completed in 15 min. With these priorities, this platform has been applied successfully to the detection of MRSA and KPC-2 KP in human urine, saliva, cerebrospinal fluid, and bovine blood. Utilizing different aptamers, this strategy could also be used to monitor other target bacteria with high specificity and sensitivity.

Versatile self-assembled MXene-Au nanocomposites for SERS detection of bacteria, antibacterial and photothermal sterilization

The emergence of antibiotic-resistant bacterial strains has caused bacterial infections to become a more serious worldwide health problem. The development of multi-functional platform that can quickly and sensitively detect bacteria and effectively inhibit or kill bacterial is important and urgent. Herein, a novel versatile MXene-Au nanocomposite was successfully synthesized by self-assembly method for rapid detection of bacteria and photothermal sterilization. The typical gram-negative Escherichia coli (E. coli) and the gram-positive Bacillus subtilis (B. subtilis) were used as models to perform label-free, rapid and sensitive detection of bacteria based on SERS method. The antibacterial performance of the material was proved by the colony counting method, and the survival rates of E. coli and B. subtilis were as low as 8.05% and 0.06%, respectively. The Ti3C2Tx-Au nanoparticles (NPs) irradiated with 808 nm light showed significant antibacterial effect within only 6 min, and the germicidal rates for E. coli and B. subtilis were 99.25% and 100%, respectively. The photothermal conversion efficiency reached as high as 43.40%. Accordingly, this multi-functional nanocomposite material can not only detect bacteria sensitively, but also has antibacterial and photothermal sterilization effects, which provides a greatly promising countermeasure for clinical treatment of diseases caused by multi-drug resistant bacteria.

Recent progress on microfluidic biosensors for rapid detection of pathogenic bacteria

Rapid on-site detection of pathogenic bacteria with high sensitivity and specificity is becoming an urgent need in public health assurance, medical diagnostics, environmental monitoring, and food safety fields. Despite being reliable and widely used, the existing methods of bacteria detection are cumbersome and time-consuming, which is not conducive to field detection. Microfluidic lab-on-a-chip technology has provided a detective tool for various analytes, due to its miniaturization, portability and low reagent consumption. Within this progress report, advances in the bacteria detection using microfluidic biosensors were discussed. Typical methods for pathogenic bacteria capture, separation and detection were introduced respectively in the first part. Then key applications of microfluidic biosensor-based rapid bacteria detection were presented. Finally, we made a conclusion and discussed possible research prospects in aspects of microfluidic biosensors for rapid detection of pathogenic bacteria.

Ultrasensitive pathogenic bacteria detection by a smartphone-read G-quadruplex-based CRISPR-Cas12a bioassay

Foodborne diseases, caused by pathogenic bacteria, severely threaten global human health and cause a financial burden. Rapid, sensitive and on-site detection of pathogenic bacteria is significant. The existing methods have different defects, such as time-consuming and inconvenient. In this study, we developed a G-quadruplex-based CRISPR-Cas12a bioassay for pathogenic bacteria detection with high sensitivity and visualization capability. Salmonella was used as the detection model. Simply, the amplicons of Salmonella specific invA gene activated the trans-cleavage activity of Cas12a and triggered CRISPR-Cas12a based indiscriminate degradation of single-stranded DNAs (ssDNAs). The ssDNAs were designed with the guanine-rich sequence and formed a stable G-quadruplex DNAzyme by adding K+. This DNAzyme could catalyze the TMB-H2O2 reaction in the presence of hemin, leading to an increase in absorbance at 454 nm and a color change. This change can be readily differentiated by the naked eyes as well as a smartphone with a Color Picker App. With this strategy, the limit of detection (LOD) for Salmonella was 1 CFU/mL with no cross-reactivity. A linear relationship (R2 = 0.993) between the absorbance and the concentration of Salmonella was obtained. Furthermore, G-quadruplex-based CRISPR-Cas12a bioassay was successfully applied for sensing Salmonella in real food samples. This work not only expands the reach of CRISPR-Cas based biosensing but also provides a novel pathogenic bacteria detection method with high sensitivity, specificity and on-site capability.

Nanozyme sensor based-on platinum-decorated polymer nanosphere for rapid and sensitive detection of Salmonella typhimurium with the naked eye

Increasingly frequent outbreaks of bacteria-caused illness heightened the need to develop a rapid, portable, sensitive, and reliable method for bacteria detection. Herein, a nanozyme Ps-Pt was synthesized by growing Pt nanoparticles (Pt) on the surface of carboxyl functionalized polymer nanospheres (Ps). The Pt endowed the Ps-Pt with ultrahigh peroxidase-like catalytic activity. Meanwhile, carboxyl groups enabled Ps-Pt to attach streptavidin via covalent binding, which showed higher activity and stability than physical adsorption. To get immune Ps-Pt (IPs-Pt), biotin-antibody was attached to the streptavidin modified Ps-Pt. The two-step binding method enabled the IPs-Pt to own more antibodies and possess high stability. Based on the immune nanozyme, a simple, sensitive colorimetric assay was reported for Salmonella typhimurium (S. typhi) detection. With the assay, as low as 100 CFU/mL S. typhi could be distinguished from the blank by measuring the absorbance, and the generated color change of 1000 CFU/mL S. typhi could be seen clearly with the naked eye. Additionally, the assay has been successfully applied to the detection of S. typhi in spiked milk, Luria-Bertani broth, and serum samples. The specificity and sensitivity of the IPs-Pt-based sensor demonstrated its feasibility in practical applications. Most importantly, the results of 24 single-blind milk samples demonstrated the high sensitivity and 100 % accuracy of the assay, indicated that the developed method could detect S. typhi in food samples.

Rapid detection and identification of bacteria directly from whole blood with light scattering spectroscopy based biosensor

Bacterial infections are one of the major causes of death worldwide. The identification of a bacterial species that is the source of an infection generally takes a long time, and often exceeds the treatment window for seriously ill patients. Many of these deaths are preventable if the bacterial species can be identified quickly. Here we present an optical spectroscopic method for rapid detection and identification of bacteria directly from whole blood using a light scattering spectroscopy technique. This technique was originally developed to detect pre-cancerous changes in epithelial tissues, characterize changes in tissue on the cellular scale, and characterize biological structures comparable to or smaller than a single wavelength. We demonstrate here that not only can an inexpensive light scattering spectroscopy-based biosensor rapidly detect and identify four bacteria species in the blood, responsible for the majority of death causing infections, but that species-level identification can potentially be made based on approximately one thousand bacterial cells per milliliter of blood. Observing entire colonies or performing susceptibility testing is therefore not required.

Quick detection of causative bacteria in cases of acute cholangitis and cholecystitis using a multichannel gene autoanalyzer.

Acute cholangitis and cholecystitis can become severe conditions as a result of inappropriate therapeutic administration and thereafter become increasingly resistant to antimicrobial treatment. The simultaneous detection of the bacterial nucleic acid and antimicrobial resistance gene is covered by the national health insurance program in Japan for sepsis. In this study, we evaluate the use of a multichannel gene autoanalyzer (Verigene system) for the quick detection of causative bacteria in cases of acute cholangitis and cholecystitis. This study included 108 patients diagnosed with acute cholangitis or cholecystitis between June 2015 and November 2018. A bacterial culture test and Verigene assay were used to evaluate the bile samples. The most commonly isolated bacteria were Escherichia coli, which includes six extended-spectrum beta-lactamase (ESBL)-producing E. coli. Among the patients with positive bile cultures, bacteria were detected in 35.7% of cases via the Verigene system. The detection rates of the Verigene system significantly increased when the number of bacterial colonies was ≥ 106 colony-forming unit (CFU)/mL (58.1%). Cases with a maximum colony quantity of ≥ 106CFU/mL exhibited higher inflammation, suggesting the presence of a bacterial infection. The Verigene system might be a new method for the quick detection of causative bacteria in patients with infectious acute cholangitis and cholecystitis.

Ultrasensitive Detection of Bacteria Using a 2D MOF Nanozyme-Amplified Electrochemical Detector.

Bacterial infection is one of the major causes of human death worldwide. To prevent bacterial infectious diseases from spreading, it is of critical importance to develop convenient, ultrasensitive, and cost-efficient methods for bacteria detection. Here, an electrochemical detector of a functional two-dimensional (2D) metal-organic framework (MOF) nanozyme was developed for the sensitive detection of pathogenic Staphylococcus aureus. A dual recognition strategy consisting of vancomycin and anti-S. aureus antibody was proposed to specifically anchor S. aureus. The 2D MOFs with excellent peroxidase-like activity can efficiently catalyze o-phenylenediamine to 2,2-diaminoazobenzene, which is an ideal electrochemical signal readout for monitoring the bacteria concentration. Under optimal conditions, the present bioassay provides a wide detection range of 10-7.5 × 107 colony-forming units CFU/mL with a detection limit of 6 CFU/mL, which is better than most of the previous reports. In addition, the established electrochemical sensor can selectively and accurately identify S. aureus in the presence of other bacteria. The present work provides a new pathway for sensitive and selective detection of S. aureus and presents a promising potential in the realm of clinical diagnosis.

Study on Molecular Qualitative Detection Technology of Potato Scab Disease

The paper uses a method that combines biological characteristics and 16S rDNA sequence characteristics to analyze potato scab bacteria collected from China. The study found that the obtained universal detection primers B1/B2 showed good specificity for all pathogenic strains, and could stably expand the target band, while non-pathogenic strains had no bands. The paper uses the spore dilution method to verify that the established detection method has a sensitivity of 20 pg·μL-1 for strain DNA, and the detection threshold for spores is about 4.0 CFU/μL. The genome amplification results of the test samples showed that the target bands were detected in the scab pathogen Streptomyces, diseased potato sample tissues, and diseased soil samples, but not the scab pathogen Streptomyces, healthy tuber tissues, non-sick soil samples and other strains None of the bands of interest were expanded, indicating that the method has good specificity. Therefore, we can conclude that the qualitative detection method of potato scab bacteria can realize rapid detection of strains, diseased tissues and soil samples.

Fast and Sensitive Bacteria Detection by Boronic Acid Modified Fluorescent Dendrimer.

This study reports a novel, fast, easy, and sensitive detection method for bacteria which is urgently needed to diagnose infections in their early stages. Our work presents a complex of poly(amidoamine) dendrimer modified by phenylboronic acid and labeled by a fluorescent dansyl group (Dan-B8.5-PAMAM). Our system detects bacteria in 20 min with a sensitivity of approximately 104 colony-forming units (CFU)·mL−1. Moreover, it does not require any peculiar technical skills or expensive materials. The driving force for bacteria recognition is the binding between terminal phenylboronic acids on the probe and bacteria’s surface glycolipids, rather than electrostatic interactions. The aggregation caused by such binding reduces fluorescence. Even though our recognition method does not distinguish between live or dead bacteria, it shows selective antibacterial activity towards Gram-negative bacteria. This study may potentially contribute a new method for the convenient detection and killing of bacteria.

Sensitive detection of foodborne pathogens based on CRISPR-Cas13a.

Salmonella, being one of the most widespread foodborne pathogens, is a compulsory test item required by national food safety standard of China and many other countries. More sensitive and specific Salmonella detection method is still needed since traditional methods are time consuming and highly dependent on enormous manpower and material resources. In this research, a bacteria detection method based on CRISPR-Cas13a system (where CRISPR is Clustered Regularly Interspaced Short Palindromic Repeats) was proposed. The target DNA was amplified by PCR and transcribed into RNA by T7 transcriptase, which can activate the RNase activity of the Cas13a protein. The self-folding quenched fluorescent probe can be cleaved by the activated Cas13a protein to generate fluorescent signal. We named this method as PCF detection (PCR-CRISPR-Fluorescence based nucleic acid detection). In this study, PCF detection showed excellent sensitivity, which can detect Salmonella genomic DNA with a minimum of 101 aM or 10° CFU/ml Salmonella bacteria in 2hr. It also showed good specificity with no cross-reaction with other common foodborne bacteria. PRACTICAL APPLICATION: The PCF detection method proposed in this article can detect Salmonella sensitively and specifically, providing a novel strategy for the detection of foodborne pathogens in food and has great application potential in other microbial detection fields.

A universal SERS-label immunoassay for pathogen bacteria detection based on Fe3O4@Au-aptamer separation and antibody-protein A orientation recognition

Rapid, reliable and sensitive detection methods for pathogenic bacteria are strongly demanded. Herein, we proposed a magnetically assisted surface enhanced Raman scattering (SERS)-label immunoassay for the sensitive detection of bacteria by using a universal approach based on free antibody labelling and staphylococcus proteins A (PA)-SERS tags orientation recognition. The SERS biosensor consists of two functional nanomaterials: aptamer-conjugated Fe3O4@Au magnetic nanoparticles (MNPs) as magnetic SERS platform for pathogen enrichment and PA modified-SERS tags (Au@DTNB@PA) as a universal probe for target bacteria quantitative detection. After target bacteria enriched, free antibody was used to specific marking target bacteria and provided numerous Fc fragment, which can guide the PA-SERS tags orientation-dependent binding. With this strategy, Fe3O4@Au/bacteria/SERS tags sandwich immunocomplexes for most bacteria (expect several species of Staphylococcus) were easy constructed. The limits of detection (LODs) of the proposed assay were found to be 10, 10, and 25 cells/mL for three common pathogens Escherichia coli (E. coli), Listeria monocytogenes (L. mono), and Salmonella typhimurium (S. typhi), respectively, in real food samples. The universal method also exhibits the advantages of rapid, robust, and easy to operate, suggesting its great potential for food safety monitoring and infectious diseases diagnosis.

Novel detection methods for dormant bacteria in seafood

Novel detection methods for dormant bacteria in seafood

DNAzyme biosensors for the detection of pathogenic bacteria

• DNAzymes can be used for the detection and diagnosis of pathogenic bacteria. • DNAzymes can be used to detect pathogenic bacteria in food and environments. • DNAzymes have potential applications in molecules detection. • RNA-cleaving DNAzymes as a molecular beacon have great advantages in fabrication of sensors. Pathogenic bacteria contamination can cause many problems, such as infections in humans and animals and environmental pollution. Early prevention and detection of pathogenic bacteria outbreaks is crucial to managing these problems. Some traditional detection methods such as cell culturing and polymerase chain reaction (PCR) are time-consuming, labor-intensive, insensitive, and inconvenient for detection. Therefore, simple and efficient pathogenic bacteria detection methods involving DNAzymes signaling mechanisms have been reported, including fluorescence and color detection. DNAzymes are divided into cases as a molecular recognition element (RNA-cleaving DNAzyme) and as a reporter element (peroxidase mimicking DNAzyme), which have proven compatible with isothermal amplification technology for pathogenic bacteria detection. In this review, we summarize the current methods of detecting pathogenic bacteria based on various DNAzyme sensors. We include the design principles, working mechanisms, and detection applications of these sensors and discuss the experimental and theoretical bases for their application in the diagnosis and prevention of diseases caused by pathogenic bacteria.

Recent advances on aptamer-based biosensors for detection of pathogenic bacteria.

As a significant constituent in biosphere, bacteria have a great influence on human activity. The detection of pathogen bacteria is closely related to the human health. However, the traditional methods for detection of pathogenic bacteria are time-consuming and difficult for quantification, although they are practical and reliable. Therefore, novel strategies for rapid, sensitive, and cost-effective detection are in great demand. Aptamer is a kind of oligonucleotide that selected by repeated screening in vitro or systematic evolution of ligands by exponential enrichment (SELEX) technology. Over the past years, owing to high affinity and specificity of aptamers, a variety of aptamer-based biosensors have been designed and applied for pathogen detection. In this review, we have discussed the recent advances on the applications of aptamer-based biosensors in detection of pathogenic bacteria. In addition, we also point out some problems in current methods and look forward to the further development of aptamer-based biosensors for pathogen detection.

Rapid, sensitive and label-free detection of pathogenic bacteria using a bacteria-imprinted conducting polymer film-based electrochemical sensor

The rapid and sensitive detection of pathogenic bacteria is very important for timely prevention and treatment of foodborne disease. Here, a bacteria-imprinted conductive poly(3-thiopheneacetic acid) (BICP) film-based impedimetric sensor was developed for the rapid, sensitive and label-free detection of staphylococcus aureus (S. aureus). The BICP film preparation was very convenient and eco-friendly, which was in situ deposited on gold electrode surface without the use of toxic organic solvents and cross-linkers. The process of imprinting and recognition were characterized by electrochemical technique and scanning electron microscope. The BICP had a novel structure without cocci-shaped cavities formed in the poly(3-thiopheneacetic acid) (PTAA) matrices. To obtain the optimal sensing performance, a set of factors affecting the imprinting and recognition were investigated. Under the optimized conditions, an extremely rapid recognition within 10 min, a very low limit of detection (LOD) of 2 CFU/mL, and wide linear range from 10 to 108 CFU/mL were achieved by the BICP film-based impedimetric sensor. The sensor also demonstrated high selectivity, and good universality and repeatability. Furthermore, the feasibility of its application has also been demonstrated in the analysis of real milk samples. This sensor offered a simple and universal method for rapid, sensitive, and selective detection of pathogenic bacteria, which could hold great potentials in fields like food safety.

Rapid Detection of Helicobacter pylori by the Naked Eye Using DNA Aptamers

Helicobacter pylori was first isolated from gastritis patients by Barry J. Marshall and J. Robin Warren in 1982, and more than 90% of duodenal ulcers and about 80% of gastric ulcers are caused by H. pylori infection. Most detection methods require sophisticated instruments and professional operators, making detection slow and expensive. Therefore, it is critical to develop a simple, fast, highly specific, and practical strategy for the detection of H. pylori. In this study, we used H. pylori as a target to select unique aptamers that can be used for the detection of H. pylori. In our study, we used random ssDNA as an initial library to screen nucleic acid aptamers for H. pylori. We used binding rate and the fluorescence intensity to identify candidate aptamers. One DNA aptamer, named HPA-2, was discovered through six rounds of positive selection and three rounds of negative selection, and it had the highest affinity constant of all aptamers tested (Kd = 19.3 ± 3.2 nM). This aptamer could be used to detect H. pylori and showed no specificity for other bacteria. Moreover, we developed a new sensor to detect H. pylori with the naked eye for 5 min using illumination from a hand-held flashlight. Our study provides a framework for the development of other aptamer-based methods for the rapid detection of pathogenic bacteria.