Food safety is one of the greatest public health challenges. Developing ultrasensitive detection methods for analytes at ultra-trace levels is, therefore, essential. In recent years, the bio-barcode assay (BCA) has emerged as an effective ultrasensitive detection strategy that is based on the indirect amplification of various DNA probes. This review systematically summarizes the progress of fluorescence, PCR, and colorimetry-based BCA methods for the detection of various contaminants, including pathogenic bacteria, toxins, pesticides, antibiotics, and other chemical substances in food in over 120 research papers. Current challenges, including long experimental times and strict storage conditions, and the prospects for the application of BCA in biomedicine and environmental analyses, have also been discussed herein.

Development of a sensitive, specific and cost-effective DNA detection method is motivated by increasing demand for the early stage diagnosis of genetic diseases. Recent developments in the design and fabrication of efficient sensor platforms based on nanostructures make the highly sensitive sensors which could indicate very low detection limit to the level of few molecules, a realistic possibility. Electrochemical detection methods are widely used in DNA diagnostics as it provide simple, accurate and inexpensive platform for DNA detection. In addition, the electrochemical DNA sensors provide direct electronic signal without the use of expensive signal transduction equipment and facilitates the immobilization of single stranded DNA (ssDNA) probe sequences on a wide variety of electrode substrates. It has been found that a range of nanomaterials such as metal nanoparticles (MNPs), carbon based nanomaterials, quantum dots (QDs), magnetic nanoparticles and polymeric NPs have been introduced in the sensor design to enhance the sensing performance of electrochemical DNA sensor. In this review, we discuss recent progress in the design and fabrication of efficient electrochemical genosensors based on carbon nanostructures such as carbon nanotubes, graphene, graphene oxide and nanodiamonds.

Only 25% of multidrug-resistant tuberculosis (MDR-TB) cases are currently diagnosed. Line probe assays (LPAs) enable rapid drug-susceptibility testing for rifampicin (RIF) and isoniazid (INH) resistance and Mycobacterium tuberculosis detection. Genotype MTBDRplusV1 was WHO-endorsed in 2008 but newer LPAs have since been developed.This systematic review evaluated three LPAs: Hain Genotype MTBDRplusV1, MTBDRplusV2 and Nipro NTM+MDRTB. Study quality was assessed with QUADAS-2. Bivariate random-effects meta-analyses were performed for direct and indirect testing. Results for RIF and INH resistance were compared to phenotypic and composite (incorporating sequencing) reference standards. M. tuberculosis detection results were compared to culture.74 unique studies were included. For RIF resistance (21 225 samples), pooled sensitivity and specificity (with 95% confidence intervals) were 96.7% (95.6-97.5%) and 98.8% (98.2-99.2%). For INH resistance (20 954 samples), pooled sensitivity and specificity were 90.2% (88.2-91.9%) and 99.2% (98.7-99.5%). Results were similar for direct and indirect testing and across LPAs. Using a composite reference standard, specificity increased marginally. For M. tuberculosis detection (3451 samples), pooled sensitivity was 94% (89.4-99.4%) for smear-positive specimens and 44% (20.2-71.7%) for smear-negative specimens.In patients with pulmonary TB, LPAs have high sensitivity and specificity for RIF resistance and high specificity and good sensitivity for INH resistance. This meta-analysis provides evidence for policy and practice.

Microbial communities are the engines that drive the global biogeochemical cycle of carbon and nitrogen essential for life on Earth. However, microorganisms have evolved as a result of complex interactions with other organisms and environments. Deciphering the metabolism of microorganisms at the community level in nature will be crucial for a better understanding of the mechanisms that lead to the enormous divergence of microbial ecophysiology. Due to the immense number of uncultivated microbial species and the complexity of microbial communities, delineating community metabolism proves a virtually insurmountable hurdle. By tracing the heavy isotope flow of key elements such as carbon and nitrogen, DNA-based stable isotope probing (DNA-SIP) can provide unequivocal evidence for substrate assimilation by microorganisms in complex environments. The essential prerequisite for a successful DNA-SIP is the identification, with confidence, of isotopically enriched 13C-DNA, of which the amount is generally too low to allow the direct measurement of 13C atom percent of nucleic acid. The methodological considerations for obtaining unambiguous DNA highly enriched in heavy isotope are presented with emphasis on next-generation sequencing technology and metagenomics.

DNA stable-isotope probing (DNA-SIP) is a recently developed method with which the incorporation of stable isotope from a labeled substrate is used to identify the function of microorganisms in the environment. The technique has now been used in conjunction with metagenomics to establish links between microbial identity and particular metabolic functions. The combination of DNA-SIP and metagenomics not only permits the detection of rare low-abundance species from metagenomic libraries but also facilitates the detection of novel enzymes and bioactive compounds. We summarize recent progress in SIP-metagenomic techniques and applications and discuss prospects for this combined approach in environmental microbiology and biotechnology.

Microarray DNA hybridization techniques have been used widely from basic to applied molecular biology research. Generally, in a DNA microarray, different probe DNA molecules are immobilized on a solid support in groups and form an array of microspots. Then, hybridization to the microarray can be performed by applying sample DNA solutions in either the bulk or the microfluidic manner. Because the immobilized probe DNA binds and retains its complementary target DNA, detection is achieved through the read-out of the tagged markers on the sample target molecules. The recent microfluidic hybridization method shows the advantages of less sample usage and reduced incubation time. Here, sample solutions are confined in microfabricated channels and flow through the probe microarray area. The high surface-to-volume ratio in microchannels of nanolitre volume greatly enhanced the sensitivity as obtained with the bulk solution method. To generate nanolitre flows, different techniques have been developed, and this including electrokinetic control, vacuum suction and syringe pumping. The latter two are pressure-driven methods which are more flexible without the need of considering the physicochemical properties of solutions. Recently, centrifugal force is employed to drive liquid movement in microchannels. This method utilizes the body force from the liquid itself and there are no additional solution interface contacts such as from electrodes or syringes and tubing. Centrifugal force driven flow also features the ease of parallel hybridizations. In this review, we will summarize the recent advances in microfluidic microarray hybridization and compare the applications of various flow methods.

The comet assay (single-cell gel electrophoresis) is now the most popular method for measuring low levels of damage in cellular DNA. Cells are embedded in agarose on a microscope slide and lysed to produce nucleoids of supercoiled DNA attached to the nuclear matrix. Breaks in the DNA relax the supercoiling and allow DNA loops to expand, and on electrophoresis to move towards the anode, giving the appearance of a comet tail. The % of DNA in the tail reflects the break frequency. Digestion of nucleoid DNA with lesion-specific endonucleases extends the usefulness of the method to investigate different kinds of damage. DNA repair can be studied by treating cells with a genotoxic agent, incubating them and using the comet assay to follow the removal of the damage. An important feature of the assay is that damage is detected at the level of individual cells. The comet assay can be combined with fluorescent in situ hybridization, using labelled probes to particular DNA sequences, and DNA damage and repair can be examined at an even finer level of resolution. Here, we provide a general review of the technique, answer some technical and theoretical questions and give examples of applications of the method.

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BACKGROUND: Certain specific bacterial species from the subgingival biofilm have demonstrated aetiological relevance in the initiation and progression of periodontitis. Among all the bacteria studied, three have shown the highest association with destructive periodontal diseases: Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf). Therefore, the relevance of having accurate microbiological diagnostic techniques for their identification and quantification is clearly justified.
OBJECTIVE: To evaluate critically all scientific information on the currently available microbial diagnostic techniques aimed for the identification and quantification of Aa, Pg and Tf.
CONCLUSIONS: Bacterial culturing has been the reference diagnostic technique for many years and, in fact, most of our current knowledge on periodontal microbiology derives from cultural data. However, the advent of new microbial diagnostics, mostly based on immune and molecular technologies, has not only highlighted some of the shortcomings of cultural techniques but has also allowed their introduction as easy and available adjunct diagnostic tools to be used in clinical research and practice. These technologies, mostly polymerase chain reaction (PCR), represent a field of continuous development; however, we still lack the ideal diagnostic to study the subgingival microflora. Qualitative PCR is still hampered by the limited information provided. Quantitative PCR is still in development; however, the promising early results reported are still hampered by the high cost and the equipment necessary for the processing.
CONCLUSIONS: Quantitative PCR technology may have a major role in the near future as an adjunctive diagnostic tool in both epidemiological and clinical studies in periodontology. However, culture techniques still hold some inherent capabilities, which makes this diagnostic tool the current reference standard in periodontal microbiology.

The gram-negative bacterium Vibrio vulnificus is a natural inhabitant of estuarine waters and poses a significant health threat to humans who suffer from immune disorders, liver disease, or hemochromatosis (iron overload). V. vulnificus enters human hosts via wound infections or consumption of raw shellfish (primarily oysters), and infections frequently progress to septicemia and death in susceptible individuals. Prevalence in waters and shellfish is not correlated with fecal indicator organisms; therefore, species-specific detection and enumeration of V. vulnificus in the environment has become a priority for agencies that are responsible for shellfish safety. The many selective-differential media developed for isolation of Vibrio spp., and specifically for V. vulnificus detection, are reviewed here; however, none of the media developed to date combines the sensitivity to low numbers with the specificity necessary to inhibit growth of other organisms. Therefore, immunological and molecular protocols are needed for confirmation of the identity of the organism and are discussed in detail. Methods under development that hold promise for rapid, accurate, and sensitive detection and enumeration of the organism include multiplex and real-time PCR. Developing technologies that have proven useful for detection and investigation of other pathogens such as biosensors, spectroscopy and microarrays may provide the next generation of tools for investigation of the prevalence and ecology of V. vulnificus.