A Rapid Digital PCR System with a Pressurized Thermal Cycler

We designed a silicon-based fast-generated static droplets array (SDA) chip and developed a rapid digital polymerase chain reaction (dPCR) detection platform that is easy to load samples for fluorescence monitoring. By using the direct scraping method for sample loading, a droplet array of 2704 microwells with each volume of about 0.785 nL can be easily realized. It was determined that the sample loading time was less than 10 s with very simple and efficient characteristics. In this platform, a pressurized thermal cycling device was first used to solve the evaporation problem usually encountered for dPCR experiments, which is critical to ensuring the successful amplification of templates at the nanoliter scale. We used a gradient dilution of the hepatitis B virus (HBV) plasmid as the target DNA for a dPCR reaction to test the feasibility of the dPCR chip. Our experimental results demonstrated that the dPCR chip could be used to quantitatively detect DNA molecules. Furthermore, the platform can measure the fluorescence intensity in real-time. To test the accuracy of the digital PCR system, we chose three-channel silicon-based chips to operate real-time fluorescent PCR experiments on this platform.

Absolute quantification of E. coli virulence and housekeeping genes to determine pathogen loads in enumerated environmental samples

Quantifying pathogenic genes with q-PCR in complex samples to determine the pathogen loads is influenced by a wide range of factors, including choice of extraction method, standard curve, and the decision to use relative versus absolute quantification of the genes. The aim was to investigate the standardisation of q-PCR methods to determine enumerated E. coli gene ratios grown with the IDEXX Colilert® Quanti-Trays® using enteropathogenic E. coli as the model pathogen. q-PCR targeting the eaeA and gadAB genes was used to calculate the eaeA: gadAB ratios for clinical strains collected between [2005–2006 (n = 55)] and [2008–2009 (n = 19)] using the LinRegPCR software and Corbett Research Thermal cycler software. Both programs grouped the isolates into two distinct groups based on the gene ratios although the Corbett Research Thermal cycler software gave results one log higher than the LinRegPCR program. Although the eaeA: gadAB ratio range was determined using extracted E. coli DNA, the impact of free DNA and other bacteria present in the sample needed to be understood. Standard curve variations using serially diluted extracted E. coli DNA, serially diluted pure E. coli culture followed by DNA extraction from each dilution with or without other bacteria was tested using the eaeA q-PCR to quantify the genes. Comparison of the standard curves showed no significant difference between standard curves prepared with diluted DNA or with cells diluted before the DNA is extracted (P = 0.435). Significant differences were observed when background DNA was included in the diluent or Coliform cells added to the diluent to dilute cells before the DNA is extracted (P < 0.001). The “carrier” DNA and Coliform cells enhanced the DNA extraction results resulting in better PCR efficiency. This will have an influence on the quantification of gene ratios and pathogen load in samples containing lower numbers of E. coli.

LAMP assays for the simple and rapid detection of clinically important urinary pathogens including the detection of resistance to 3rd generation cephalosporins

Background Timely and accurate identification of uropathogens and determination of their antimicrobial susceptibility is paramount to the management of urinary tract infections (UTIs). The main objective of this study was to develop an assay using LAMP (Loop mediated isothermal amplification) technology for simple, rapid and sensitive detection of the most common bacteria responsible for UTIs, as well as for the detection of the most prevalent genes (encoding cefotaximases from CTX-M group 1) responsible for resistance to 3rd generation of cephalosporins. Method We designed primers targeting Proteus mirabilis, while those targeting Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis and the CTX-M group 1 resistance gene were benchmarked from previous studies. The amplification reaction was carried out in a warm water bath for 60 min at 63 ± 0.5 °C. The amplicons were revealed by staining with Sybr Green I. Specificity and sensitivity were determined using reference DNA extracts spiked in sterile urine samples. The analytical performance of the assays was evaluated directly on pellets of urine samples from patients suspected of UTI and compared with culture. Results We found a high specificity (100%) for LAMP assays targeting the selected bacteria (P. mirabilis, E. coli, K. pneumoniae, E. faecalis) and the CTX-M group 1 when using DNA extracts spiked in urine samples. The sensitivities of the assays were around 1.5 103 Colony Forming Units (CFU) /mL corresponding to the cut-off value used to define bacteriuria or UTIs in patients with symptoms. Out of 161 urine samples tested, using culture as gold standard, we found a sensitivity of the LAMP techniques ranging from 96 to 100% and specificity from 95 to 100%. Conclusion We showed that the LAMP assays were simple and fast. The tests showed high sensitivity and specificity using a simple procedure for DNA extraction. In addition, the assays could be performed without the need of an expensive device such as a thermal cycler. These LAMP assays could be useful as an alternative or a complementary tool to culture reducing the time to diagnosis and guiding for more effective treatment of UTIs but also as a powerful diagnostic tool in resource-limited countries where culture is not available in primary health care structures.

In-house reverse transcriptase polymerase chain reaction for detection of SARS-CoV-2 with increased sensitivity

As the COVID-19 infection continues to ravage the world, the advent of an efficient as well as the economization of the existing RT-PCR based detection assay essentially can become a blessing in these testing times and significantly help in the management of the pandemic. This study demonstrated an innovative and rapid corroboration of COVID-19 test based on innovative multiplex PCR. An assessment of optimal PCR conditions to simultaneously amplify the SARS-CoV-2 genes E, S and RdRp has been made by fast-conventional and HRM coupled multiplex real-time PCR using the same sets of primers. All variables of practical value were studied by amplifying known target-sequences from ten-fold dilutions of archived positive samples of COVID-19 disease. The multiplexing with newly designed E, S and RdRp primers have shown an efficient amplification of the target region of SARS-CoV-2. A distinct amplification was observed in 37 min using thermal cycler while it took 96 min in HRM coupled real time detection using SYBR green over a wide range of template concentrations. Our findings revealed decent concordance with other commercially available detection kits. This fast HRM coupled multiplex real-time PCR with SYBR green approach offers rapid and sensitive detection of SARS-CoV-2 in a cost-effective manner apart from the added advantage of primer compatibility for use in conventional multiplex PCR. The highly reproducible novel approach can propel extended applicability for developing sustainable commercial product besides providing relief to a resource limited setting.

Morphological and Molecular Characterization of Alternaria Species Isolated from Different Plants

Alternaria belongs to the family of Pleosporaceae, the order of Pleosporales, the class of Dothideomycetes in the phyllum of Ascomycota. This fungal genus is characterized by its ability to produce a number of enzymes able to injured many types of crops. The symptoms of Alternaria blight on different agricultural crops such as cabbage, cauliflower, coriander, fenugreek, brinjal, onion, faba bean, dahlia, dracaena, hollyhock, carrot, marigold, tomato and wheat were observed. Alternaria produces distinctive "bulls eye" patterned leaf spots in almost all the infected plants. Nineteen Alternaria isolates were obtained from fourteen infected plant samples. The observations were recorded from the seven day old culture for colony characteristics on PDA. Based on morphology 10 isolates of Alternaria alternata, two isolates Alternaria tenuissima and Alternaria longipes and one Alternaria porri, Alternaria brassicae, Alternaria brassicicola and Alternaria sp. were identified. Alternaria species grow rapidly and produced flat, downy to woolly grayish green to black colonies. All the isolates exhibited characteristics dark-coloured multicelled conidia with longitudinal and transverse septa (phaeo-dictyospores) and a beak or tapering apical cells. DNA isolation of all isolates of Alternaria was carried out using CTAB method. Isolated DNA was subjected to amplification with ITS1 and ITS4 primers in a thermal cycler. The amplified products ranged from 580-600bp. These amplified products were sequenced and identify the species of different Alternaria isolates using BLASTn in NCBI online. All the sequences were published in NCBI public domain. The resulting sequences of all isolates were compared to other sequences in the GenBank as 90-100% identical. Genetic variability was conducted by phylogenetic analysis.

Polymorphism of genes CAST, GH, GDF9 of sheep of the Dagestan mountain breed

Aim. To study the polymorphism of the genes calpastatin (CAST), somatotropin (GH) and differential growth factor (GDF9) of sheep of the Dagestan mountain breed.Material and Methods. Studies on the genetic typing of Dagestan mountain sheep according to CAST, GH and GDF9 genes were carried out in conditions of distant pasture farming in the Republic of Dagestan by the Genetics and Breeding Laboratory of the Federal Agrarian Scientific Centre of the Republic of Dagestan, an accredited laboratory of immunogenetics and DNA technologies of the All-Russian Research Institute of Sheep and Goat Breeding and a branch of the North Caucasus Federal Scientific Agrarian Centre. These investigations were undertaken by the PCR-RFLP method (polymyrase chain reaction - restriction fragment length polymorphism) on a Tersik four-channel programmable thermal cycler from DNA-Technologia (Russia) using specific primers synthesized in the SYNTHOL research and production laboratory (Moscow).Results. Carrying out PCR-RFLP revealed breed-specific alleles with different frequency of occurrence, which was: in the locus of the CAST gene - allele CASTN - 0.03; allele CASTM - 0.97; at the GH gene locus - GHA allele - 100.0, GHB allele - 0; in the locus of the GDF9 gene - the GDF9A allele - 0.25, the GDF9G allele - 0.75, which determined the frequency of occurrence of the homo- and heterozygous genotypes: CASTNN, CASTMM and CASTNM - 93.0; 0 and 7.0%; GHAA, GHBB and GHAB - 100.0 and 0%; GDF9AA, GDF9GG and GDF9АG- 16.0, 66.0 and 19.0%, respectively.Conclusion. The regularity revealed can be considered as an ecological factor that optimizes the adaptive functions of the sheep's organism, on the one hand, an evolutionary selection process that contributes to the creation of a specific way of genetic structures in the population, on the other. The assumption is made that the information obtained can serve as the beginning of a more in-depth study of the unique gene pool of sheep of the Dagestan rock breed for its further improvement. Variants of genetic marker profiles of parental pairs are proposed for accumulation in breeding herds bred in different ecological-geographical zones and genotypes of carriers of selection-significant genetic structures.

One-Day Molecular Detection of Salmonella and Campylobacter in Chicken Meat: A Pilot Study

Salmonella and Campylobacter ssp. are bacterial pathogens responsible for most foodborne infections in EU countries. Poultry serves as a reservoir for these pathogens, and its important role in the meat industry makes it essential to develop a rapid detection assay able to provide results in one day. Indeed, the rapid identification of foodborne pathogens is an important instrument for the monitoring and prevention of epidemic outbreaks. To date, Salmonella and Campylobacter screening is mainly conducted through molecular methods (PCR or real-time PCR) performed after 18–24 h long enrichments. In this study, we evaluated short enrichments (0, 2, 4, and 6 h) combined with a colorimetric loop-mediated isothermal AMPlification (LAMP) or real-time PCR to detect Salmonella and Campylobacter in poultry meat contaminated at different concentration levels (101, 103, and 105 CFU/g). Our results show that real-time PCR allows the detection of Salmonella and Campylobacter, even after shorter enrichment times than prescribed by ISO references; particularly, it detected Salmonella down to 101 CFU/g since T0 and Campylobacter from 103 CFU/g since T0. Detection with LAMP was comparable to real-time PCR without the requirement of a thermal cycler and with shorter execution times. These characteristics make colorimetric LAMP a valid alternative when one-day results are needed, improving the timely identification of positive meat batches, even in the absence of specialized instrumentation.

Title: LAMP Assays for the Simple and Rapid Detection of Clinically Important Urinary Pathogens Including the Detection of Resistance to 3rd Generation Cephalosporins

Background: Timely and accurate identification of uropathogens and determination of their antimicrobial susceptibility is paramount to the management of urinary tract infections (UTIs). The main objective of this study was to develop an assay using LAMP (Loop mediated isothermal amplification) technology for simple, rapid and sensitive detection of the most common bacteria responsible for UTIs, as well as for the detection of the most prevalent genes (encoding cefotaximases from CTX-M group 1) responsible for resistance to 3rd generation of cephalosporins. Method: We designed primers targeting Proteus mirabilis, while those targeting Escherichia coli, Klebsiella pneumoniae and Enterococcus faecalis and the CTX-M group 1 resistance gene were benchmarked from previous studies. The amplification reaction was carried out in a warm water bath for 60 min at 63±0.5 °C. The amplicons were revealed by staining with Sybr Green I. Specificity and sensitivity were determined using reference DNA extracts spiked in sterile urine samples. The analytical performance of the assays was evaluated directly on pellets of urine samples from patients suspected of UTI and compared with culture.Results: We found a high specificity (100%) for LAMP assays targeting the selected bacteria (P. mirabilis, E. coli, K. pneumoniae, E. faecalis) and the CTX-M group 1 when using DNA extracts spiked in urine samples. The sensitivities of the assays were around 1.5 103 Colony Forming Units (CFU) /mL corresponding to the cut-off value used to define bacteriuria or UTIs in patients with symptoms. Out of 161 urine samples tested, using culture as gold standard, we found a sensitivity of the LAMP techniques ranging from 96 to 100 % and specificity from 95 to 100 %.Conclusion: We showed that the LAMP assays were simple and fast. The tests showed high sensitivity and specificity using a simple procedure for DNA extraction. In addition, the assays could be performed without the need of an expensive device such as a thermal cycler. These LAMP assays could be useful as an alternative or a complementary tool to culture reducing the time to diagnosis and guiding for more effective treatment of UTIs but also as a powerful diagnostic tool in resource-limited countries where culture is not available in primary health care structures.