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ABSTRACT Sputum collection and testing for tuberculosis (TB) have been problematic due to the potential for aerosolization, difficulty in generating a quality sample, and complex DNA extraction methods. Tongue swabs are inexpensive, minimally invasive, and a promising alternative to sputum collection. We investigated the diagnostic accuracy of tongue swabs for TB diagnosis using the Truenat MTB Plus assay from Molbio Diagnostics with a direct to PCR processing method. Four tongue swabs were collected per participant using two nylon-flocked swabs and two spun polyester swabs. Following tongue swab sample collection, participants also provided two sputum samples, which were tested by Cepheid Xpert MTB/RIF Ultra or culture. Of the 81 participants enrolled, 24 (30%) were positive for TB disease by sputum Ultra. Using the Truenat MTB Plus test, tongue swabs had 54% (52/96) sensitivity and 99% (218/220) specificity compared to sputum Ultra. Crude lysate was also tested using an in-house qPCR assay, which allowed for increased sample input. Using this method, tongue swabs had 70% (67/96) sensitivity and 94% (216/224) specificity. Mycobacterium tuberculosis (MTB) sample quantification using digital PCR yielded between 20 copies (minimum) and 34,000 copies (maximum) of MTB per swab. In addition, serially collected tongue swabs resulted in similar levels of detected MTB, and spun polyester swabs performed equivalently to nylon-flocked swabs. Overall, this study demonstrates that tongue swab samples are compatible with the Truenat MTB testing platform and that a direct to PCR method is a viable diagnostic solution. IMPORTANCE Tuberculosis (TB) remains one of the world’s leading infectious disease killers, despite available treatments. Although highly sensitive molecular diagnostics are available, expensive equipment and poor infrastructure have hindered their implementation in low-resource settings. Furthermore, the collection of sputum poses challenges as it is difficult for patients to produce and creates dangerous aerosols. This manuscript explores tongue swabs as a promising alternative to sputum collection. While previous studies have explored the sensitivity of tongue swabs as compared to sputum, existing literature has not addressed the need to standardize and simplify laboratory processing for easy implementation in high TB burden areas. This manuscript provides the first evidence that detection of TB from a tongue swab is possible without the use of DNA extraction or purification steps. The data provided in this manuscript will improve the collection and testing of tongue swabs for the diagnosis of TB disease.

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies. [ABSTRACT FROM AUTHOR]

Studying the entire virus replication cycle of SARS-CoV-2 is essential to identify the host factors involved and treatments to combat infection. Quantification of released virions often requires lengthy procedures, whereas quantification of viral RNA in supernatant is faster and applicable to clinical isolates. Viral RNA purification is expensive in terms of time and resources, and is often unsuitable for high-throughput screening. Direct lysis protocols were explored for patient swab samples, but the lack of virus inactivation, cost, sensitivity, and accuracy is hampering their application and usefulness for in vitro studies. Here, we show a highly sensitive, accurate, fast, and cheap direct lysis RT-qPCR method for quantification of SARS-CoV-2 in culture supernatant. This method inactivates the virus and permits detection limits of 0.043 TCID50 virus and <1.89 copy RNA template per reaction. Comparing direct lysis with RNA extraction, a mean difference of +0.69 ± 0.56 cycles was observed. Application of the method to established qPCR methods for RSV (-ve RNA), IAV (segmented -ve RNA), and BHV (dsDNA) showed wider applicability to other enveloped viruses, whereby IAV showed poorer sensitivity. This shows that accurate quantification of SARS-CoV-2 and other enveloped viruses can be achieved using direct lysis protocols, facilitating a wide range of high- and low-throughput applications. [ABSTRACT FROM AUTHOR]

Routine cell culture reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) gene expression analysis is limited in scalability due to minimum sample requirement and multistep isolation procedures. In this study, we aimed to optimize and apply a cost-effective and rapid protocol for directly sampling gene expression data from microplate cell cultures. The optimized protocol involves direct lysis of microplate well population followed by a reduced thermocycler reaction time one-step RT-qPCR assay. In applications for inflammation and stress-induced cell-based models, the direct lysis RT-qPCR microplate assay was utilized to detect IFN1 and PPP1R15A expression by poly(I:C) treated primary fibroblast cultures, IL6 expression by poly(I:C) iPSC-derived astrocytes, and differential PPP1R15A expression by ER-stressed vanishing white-matter disease patient induced pluripotent stem cell (iPSC)-derived astrocytes. In application for neural differentiation medium recipe optimizations, conditions were screened for SYN1 and VGLUT1 in neuronal cultures, and S100B, GFAP and EAAT1 in astrocyte cultures. The protocol provides microplate gene expression results from cell lysate to readout within ~35 min, with comparable cost to routine RT-qPCR, and it may be utilized to support laboratory cell-based assays in basic and applied scientific and medical fields of research including stem-cell differentiation, cell physiology, and drug mechanism studies.

RT-qPCR is a widely used method to detect miRNA expression. Compared with mRNA, miRNA has a shorter length and lower abundance which hinders the acquisition of reliable results. Thus, miRNA detection requires a method with high sensitivity and accuracy. Collecting large amounts of material is particularly difficult for oocytes and pre-implantation embryos of domestic animals. Establishing a set of miRNA detection methods that are suitable to detect trace amounts of such materials is urgently needed. In this study, the total RNA in 50 germinal vesicle (GV) oocytes was isolated through direct lysis and by using mirVana miRNA Isolation Kit and miRNeasy Micro Kit. The OD260/280 values and concentrations of the RNA in these three groups were compared to identify a superior RNA isolation method. In addition, the specificity and sensitivity of common DNA and LNA primers were compared by real-time quantitative polymerase chain reaction for miRNA detection. Results show that the RNA concentration of in the direct lysis group was significantly higher than that in the other two groups. The specificity between the DNA primers and LNA primers was identical, whereas the sensitivity of LNA primers was superior to that of DNA primers. These results suggest that direct lysis combined with LNA primers might be a suitable protocol for the miRNA detection of a small amounts of GV oocytes and pre-implantation embryos in cattle. [ABSTRACT FROM AUTHOR]

The bacteria Escherichia coli and the yeast Saccharomyces cerevisiae are currently the two most important organisms in synthetic biology. E. coli is almost always used for fundamental DNA manipulation, while yeast is the simplest host system for studying eukaryotic gene expression and performing large-scale DNA assembly. Yeast expression studies may also require altering the chromosomal DNA by homologous recombination. All these studies require the verification of the expected DNA sequence, and the fastest method of screening is colony PCR, which is direct PCR of DNA in cells without prior DNA purification. Colony PCR is hampered by the difficulty of releasing DNA into the PCR mix and by the presence of PCR inhibitors. We hereby present one protocol for E. coli and two protocols for S. cerevisiae differing in efficiency and complexity as well as an overview of past and possible future developments of efficient S. cerevisiae colony PCR protocols., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The interest to analyze single and few cell samples is rapidly increasing. Numerous extraction protocols to purify nucleic acids are available, but most of them compromise severely on yield to remove contaminants and are therefore not suitable for the analysis of samples containing small numbers of transcripts only. Here,we evaluate 17 direct cell lysis protocols for transcript yield and compatibility with downstream reverse transcription quantitative real-time PCR. Four endogenously expressed genes are assayed together with RNA and DNA spikes in the samples. We found bovine serum albumin (BSA) to be the best lysis agent, resulting in efficient cell lysis, high RNA stability, and enhanced reverse transcription efficiency. Furthermore, we found direct cell lysis with BSA superior to standard column based extraction methods, when analyzing from 1 up to 512 mammalian cells. In conclusion, direct cell lysis protocols based on BSA can be applied with most cell collection methods and are compatible with most analytical workflows to analyze single-cells as well as samples composed of small numbers of cells. [ABSTRACT FROM AUTHOR]

There is an increased interest in the extraction of nucleic acids from various environmental samples, since molecular techniques allow less biased access to a greater portion of uncultivable microorganisms. Two strategies have been developed to improve DNA recovery in terms of yield, purity and unbiased representation of the microbial diversity. The first approach consists of the direct extraction of nucleic acids from soil through in situ cell lysis followed by DNA purification. The alternative approach is based on the separation of bacteria from the soil particles followed by cell lysis and then DNA purification. Several published methods describe the recovery of highly purified nucleic acids that are well-suited for molecular purposes even though a new challenge concerns the recovery of large bacterial DNAs essential for functional investigation of gene clusters and biosynthetic pathways. This review presents an overview of the available methods to achieve this challenging objective. [Copyright &y& Elsevier]

Isolation and identification of Taylorella equigenitalis , the causative agent of contagious equine metritis, by bacteriology is laborious and does not permit differentiation from the other member of the genus, Taylorella asinigenitalis. Moreover, other organisms such as Klebsiella pneumoniae and Pseudomonas aeruginosa can also cause endometritis in mares and warrant diagnostic detection. Our objectives were to develop a rapid preparation method for field swab samples and to validate this protocol using new multiplex real-time polymerase chain reaction (rtPCR) detection tools for identification of these four pathogens. The complete analytical process from sample preparation to PCR analysis was then evaluated against bacteriology, the World Organisation for Health's (OIE) gold standard method for T. equigenitalis and commonly used for the other three pathogens. The diagnostic sensitivity and specificity of this method, which used direct lysis and a multiplex rtPCR, were 100% and >92%, respectively. This study provided a simple-to-use method for prebreeding screening of mares and stallions. • New multiplex real-time PCR for rapid detection of CEM and other organisms associated with endometritis in mares. • Combination of direct lysis and PCR as rapid screening tool from swabs and semen. • Simple-to-use method for prebreeding screening of mares and stallions. [ABSTRACT FROM AUTHOR]

• Enhanced identification of single skin flakes through illumination and staining. • Sampling of small areas of adhesive film is favorable over single skin flake collection. • Minimized mixture creation through small area sampling technique. • Direct lysis superior to common DNA extraction methods for micro trace analysis. Many challenges are encountered in the analysis of micro traces such as touch DNA or telogen hair samples. Although DNA typing methods have become immensely more sensitive over the last years, recovery of the minute amounts of biological material in micro traces requires further enhancement. For example felony cases, where an offender oftentimes only contributes minor amounts of touch DNA, separation of victim and offender DNA poses difficulties. Whereas complete sampling of evidence generates admixed profiles, single particle collection is labor- and time-intensive. Besides optimization of identification and collection of bio particles during this study, a novel sampling strategy for enhanced DNA yield as well as success rate but simultaneous avoidance of mixture creation is proposed for tapings in forensic casework. Improvement of another crucial step in micro trace analysis, DNA extraction, involved evaluation of efficiency and DNA recovery of different extraction methods, namely magnetic bead based Maxwell extraction, Chelex, Casework Direct Kit and Investigator Casework GO! kit. Direct lysis approaches seemed to be most suitable for low template traces. Recently developed commercial kits even allow the presence of inhibitors. Improvement of embraced aspects successfully facilitates processing of micro traces in terms of time and labor. [ABSTRACT FROM AUTHOR]

Escherichia coli and Saccharomyces cerevisiae are currently the two most important organisms in synthetic biology. E.coli is almost always used for fundamental DNA manipulation while yeast is the simplest host system for studying eukaryotic gene expression and performing large scale DNA assembly. Yeast expression studies may also require altering of the chromosomal DNA by homologous recombination. All these studies require the verification of the expected DNA sequence and the fastest method of screening is colony PCR, which is direct PCR of DNA in cells without prior DNA purification. Colony PCR is hampered by the difficulty of releasing DNA into the PCR mix and the presence of PCR inhibitors. We hereby present one protocol for E. coli and two protocols for S. cerevisiae differing in efficiency and complexity as well as an overview of past and possible future developments of efficient S. cerevisiae colony PCR protocols.