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Abstract Although DNA extraction is a crucial step before polymerase chain reaction (PCR), attempts to perform PCR without DNA isolation have been ongoing since the 1990s. While partial success has been achieved with direct conventional PCR, the DNA isolation step has remained indispensable for real-time PCR. Here, we developed a method that does not require complete DNA isolation by lysing EDTA-treated whole blood samples through the application of osmotic pressure and heat, followed by centrifugation to get a clear lysate. Blood samples were mixed with distilled water, incubated at 95 °C for 20 min before centrifugation at 14,000 rpm for 5 min. The resulting lysates were used as templates for real-time PCR. Real-time PCRs were performed under identical conditions with lysates and DNA samples using 9 different primer sets. Target genes were successfully amplified using 1:10 and 1:5 diluted blood lysates both at 60 °C and 61 °C. The PCR efficiency for ACTB and PIK3CA differed by 20% and 14%, respectively, between the DNA samples and blood lysates. We successfully amplified all selected genes using “GG-RT PCR”, greater temperature, greater speed method, without the need for additional enzymes or buffers. GG-RT PCR presents a cost effective option for applications such as SNP analysis and deletion detection if appropriate primer designs are made.

Objective To establish a non-invasive single nucleotide polymorphism genotyping technology based on direct TaqMan-PCR for high-throughput genotyping of site rs688 and rs964184 associated with lipid metabolism to meet the need for early screening of at-risk populations. Methods Extracted DNA was used to optimize the PCR annealing extension temperature and amplification procedure for the designed TaqMan probe; the oral swabs were placed into the sample treatment solution and then briefly centrifuged, and a small amount of the supernatant was taken for the direct qPCR amplification analysis; the freeze-thawing stability of probe and the effects of sample storage time on genotyping results were explored. Results The technology requires only simple treatment of oral swab for PCR analysis and can be stored at room temperature for 4 d. The optimized method was able to distinguish homozygous wild type, homozygous mutant type and heterozygote at rs688 and rs964184 loci. Conclusions A fast, convenient, high-throughput, and low-cost SNP genotyping method has been established, providing an efficient and accurate new approach for large-scale screening of high-risk populations carrying susceptibility genes.

Abstract Background Culex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay. Results Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55–80%. Conclusions This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario.

Background: Culex pipiens L. is a principal vector of zoonotic arboviruses in Europe, acting in both an amplification role in enzootic transmission between avian hosts and as a bridge vector between avian hosts and mammals. The species consists of two forms which are indistinguishable using morphological methods but possess varying ecological and physiological traits that influence their vector capacity. In this study we validate methods that can be used to extract trace DNA from single pupal exuviae of Cx. pipiens for use in molecular speciation of samples. These DNA extraction methods are compared using measurement of the total yield and successful identification using a real-time polymerase chain reaction (PCR) assay. Results: Genomic DNA was initially extracted from colony-derived individuals using an ethanol precipitation method, two commercially available DNA extraction kits: DNeasy® Blood & Tissue Kit (Qiagen, UK) and Wizard® SV Genomic DNA Purification System (Promega, UK) and a direct real-time PCR method. Time elapsed between eclosion and processing of pupae significantly influenced Cx. pipiens form identification as nucleic acid concentration and PCR amplification success decreased with increased time elapsed. Real-time PCR amplification success, however, was not shown to vary significantly between the three extraction methods, with all methods successfully identifying all samples, but the direct real-time PCR method achieved a lesser amplification success rate of 70% (n = 20 for each treatment). More variable results were produced when field-derived exuviae were used, with no significant difference in real-time PCR amplification success found across the four methods and a lower overall rate of successful identification of 55–80%. Conclusions: This study shows that both colony and field derived Cx. pipiens pupal exuviae can be a useful non-invasive source of trace DNA permitting accurate biotype differentiation for at least twenty-four hours post-eclosion. The significance and utility of this technique in ecological and behavioural studies of Cx. pipiens is discussed and recommendations made for use according to experimental scenario. [ABSTRACT FROM AUTHOR]

Objective To establish a non-invasive single nucleotide polymorphism genotyping technology based on direct TaqMan-PCR for high-throughput genotyping of site rs688 and rs964184 associated with lipid metabolism to meet the need for early screening of at-risk populations. Methods Extracted DNA was used to optimize the PCR annealing extension temperature and amplification procedure for the designed TaqMan probe; the oral swabs were placed into the sample treatment solution and then briefly centrifuged, and a small amount of the supernatant was taken for the direct qPCR amplification analysis; the freeze-thawing stability of probe and the effects of sample storage time on genotyping results were explored. Results The technology requires only simple treatment of oral swab for PCR analysis and can be stored at room temperature for 4 d. The optimized method was able to distinguish homozygous wild type, homozygous mutant type and heterozygote at rs688 and rs964184 loci. Conclusions A fast, convenient, high-throughput, and low-cost SNP genotyping method has been established, providing an efficient and accurate new approach for large-scale screening of high-risk populations carrying susceptibility genes. [ABSTRACT FROM AUTHOR]

Background: Nucleic acid amplification testing is the gold standard for SARS-CoV-2 diagnostics, although it may produce a certain number of false positive results. There has not been much published about the characteristics of false positive results. In this study, based on retesting, specimens that initially tested positive for SARS-CoV-2 were classified as true or false positive groups to characterize the distribution of cycle threshold (CT) values for N1 and N2 targets and number of targets detected for each group. Methods: Specimens that were positive for N-gene on retesting and accompanied with S-gene were identified as true positives (true positive based on retesting, rTP), while specimens that retested negative were classified as false positives (false positive based on retesting, rFP). Results: Of the specimens retested, 85/127 (66.9%) were rFP, 16/47 (34.0%) specimens with both N1 and N2 targets initially detected were rFP, and the CT values for each target was higher in rFP than in rTP. ROC curve analysis showed that optimal cutoff values of CT to differentiate between rTP and rFP were 34.8 for N1 and 33.0 for N2. With the optimal cutoff values of CT for each target, out of the 24 specimens that were positive for both N1 and N2 targets and classified as rTP, 23 (95.8%) were correctly identified as true positives. rFP specimens had a single N1 target in 52/61 (85.2%) and a single N2 target in 17/19 (89.5%). Notably, no true positive results were obtained from any specimens with only N2 target detected. Conclusions: These results suggest that retesting should be performed for positive results with a CT value greater than optimal cutoff value for each target or with a single N1 target amplified, considering the possibility of a false positive. This may provide guidance on indications to perform retesting to minimize the number of false positives. [ABSTRACT FROM AUTHOR]

Background: Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350 bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes. Methods and results: The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed – from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR. Conclusions: The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination. [ABSTRACT FROM AUTHOR]

Potato viruses PLRV, PVY, PVM, PVA, PVX and PVS can cause up to 90% loss of potato harvest. Therefore, they are monitored by law in many countries using DAS-ELISA or conventional real-time RT-qPCR. Previously, we developed a multiplex real-time DiRT-PCR (Direct reverse transcript – polymerase chain reaction), which works directly on diluted tuber sap and thus saves time and chemical processing for RNA extraction or time and space in the glasshouse. So far, this method only ran on sap of single tubers which is not practical for routine testing. We are now able to sensitively test for the presence of six viruses in two multiplex reactions using the real-time DiRT-PCR on pooled samples of ten tubers. Here we show that there is an "almost perfect" agreement (Gwet's AC1 index) comparing this multiplex real-time DiRT-PCR on pooled samples with DAS-ELISA and a commercial RT-qPCR kit with a rapid extraction method. The multiplex real-time DiRT-PCR is now ready to be used for routine testing. [ABSTRACT FROM AUTHOR]

Abstract Background B chromosomes are classified as dispensable genomic components tolerated by cells, which are transmitted to progeny despite providing no benefit in most cases. They have been observed in over 2800 species of plants, animals and fungi, including numerous maize accessions. As maize is one of the most important crops worldwide, research on the maize B chromosome has been pioneering in the field. The characteristic of the B chromosome is its irregular inheritance. This results in offspring with a different number of B chromosomes compared to the parents. However, the exact number of B chromosomes in the studied plants is a crucial piece of information. Currently, assessing the number of B chromosomes in maize largely depends on cytogenetic analyses, which are laborious and time-consuming. We present an alternative approach based on the droplet digital PCR technique (ddPCR), which is faster, more efficient and provides the results within one day with the same level of accuracy. Results In this study, we report a rapid and straightforward protocol for determining the number of B chromosomes in maize plants. We developed a droplet digital PCR assay using specific primers and a TaqMan probe for the B-chromosome-linked gene and a single-copy reference gene on maize chromosome 1. The performance of the assay was successfully verified by comparison with the results of cytogenetic analyses performed in parallel. Conclusions The protocol significantly improves the efficiency of B chromosome number assessment in maize compared to cytogenetic approaches. The assay has been developed to target conserved genomic regions and can therefore be applied to a wide range of diverged maize accessions. This universal approach can be modified for chromosome number detection in other species, not only for the B chromosome but also for any other chromosome in aneuploid constitution.

Taq DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing Taq DNA polymerase for routine use in laboratories. We developed a method using Escherichia coli (E. coli) that expresses thermostable Taq DNA polymerase directly in the PCR without purification. The Taq gene was transformed into E. coli and expressed. After overnight incubation and washing, E. coli-expressing Taq DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification. [ABSTRACT FROM AUTHOR]

DNA extraction is a crucial step in all nucleic acid-based protocols to identify microorganisms. Lactic acid bacteria are a significant part of healthy microbiota in the human gastrointestinal tract. These gram-positive bacteria have several layers of peptidoglycan in the cell walls. These structures cause difficulties in the cell lysis and obtaining reliable protocols for DNA isolations. The purpose of this study was to assess the autoclave and lysozyme-based DNA purification approaches for achieving the high-quality genomic DNAs of Lactobacillus acidophilus bacteria. DNA concentrations and qualities were also compared with the commercial kit. The results showed that the proper DNA isolation methods were various, according to the downstream applications. Protocols that included lysozyme produced a higher amount of DNA than the autoclave method. Lysozyme treatment combined with silica-guanidinethiocyanate procedure was the efficient protocol with affordable cost for routine lysis of L. acidophilus bacteria. Appropriate DNA concentration and quality were obtained through this method comparable to those of the commercial kit. Inversely, autoclave treatment had little effect on the breakage of the cell walls indicating low concentrations of extracted DNAs. This method could not completely break down all the bacterial cell walls. However, the breakage of low numbers of cell walls was microscopically observed in the supernatant of the autoclaved cell suspension. The quality of this protocol was found to be adequate for performing direct polymerase chain reaction (PCR) assay on samples with large amounts of lactobacilli. These conclusions suggest attentively selecting the DNA extraction method based on the planned downstream analysis of PCR products.

Background: B chromosomes are classified as dispensable genomic components tolerated by cells, which are transmitted to progeny despite providing no benefit in most cases. They have been observed in over 2800 species of plants, animals and fungi, including numerous maize accessions. As maize is one of the most important crops worldwide, research on the maize B chromosome has been pioneering in the field. The characteristic of the B chromosome is its irregular inheritance. This results in offspring with a different number of B chromosomes compared to the parents. However, the exact number of B chromosomes in the studied plants is a crucial piece of information. Currently, assessing the number of B chromosomes in maize largely depends on cytogenetic analyses, which are laborious and time-consuming. We present an alternative approach based on the droplet digital PCR technique (ddPCR), which is faster, more efficient and provides the results within one day with the same level of accuracy. Results: In this study, we report a rapid and straightforward protocol for determining the number of B chromosomes in maize plants. We developed a droplet digital PCR assay using specific primers and a TaqMan probe for the B-chromosome-linked gene and a single-copy reference gene on maize chromosome 1. The performance of the assay was successfully verified by comparison with the results of cytogenetic analyses performed in parallel. Conclusions: The protocol significantly improves the efficiency of B chromosome number assessment in maize compared to cytogenetic approaches. The assay has been developed to target conserved genomic regions and can therefore be applied to a wide range of diverged maize accessions. This universal approach can be modified for chromosome number detection in other species, not only for the B chromosome but also for any other chromosome in aneuploid constitution. [ABSTRACT FROM AUTHOR]

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively--both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1--B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study. [ABSTRACT FROM AUTHOR]

Recent advances in new molecular biology methods and next-generation sequencing (NGS) technologies have revolutionized metabarcoding studies investigating complex microbial communities from various environments. The inevitable first step in sample preparation is DNA extraction which introduces its own set of biases and considerations. In this study, we assessed the influence of five DNA extraction methods [B1: phenol/chloroform/isoamyl extraction, B2 and B3: isopropanol and ethanol precipitations, respectively—both modifications of B1, K1: DNeasy PowerWater Kit (QIAGEN), K2: modified DNeasy PowerWater Kit (QIAGEN) and direct PCR approach (P) that completely circumvents this step on community composition and DNA yield of mock and marine sample communities from the Adriatic Sea]. B1–B3 methods generally produced higher DNA yields and more similar microbial communities, but with higher interindividual variability. Each method demonstrated significant differences in a specific community structure, where rare taxa seem to play a crucial role. There was not one superior method closest to the theoretically expected mock community composition, they all demonstrated skewed ratios, but in a similar way which might be attributed to other factors, such as primer bias or 16S rRNA gene count for specific taxa. Direct PCR represents an interesting approach when high throughput in sample processing is required. We emphasize the importance of making a cautious decision about the choice of the extraction method or direct PCR approach, but even more importantly its consistent application throughout the study.

DNA extraction is a crucial step in all nucleic acid-based protocols to identify microorganisms. Lactic acid bacteria are a significant part of healthy microbiota in the human gastrointestinal tract. These gram-positive bacteria have several layers of peptidoglycan in the cell walls. These structures cause difficulties in the cell lysis and obtaining reliable protocols for DNA isolations . The purpose of this study was to assess the autoclave and lysozyme-based DNA purification approaches for achieving the high-quality genomic DNAs of Lactobacillus acidophilus bacteria. DNA concentrations and qualities were also compared with the commercial kit. The results showed that the proper DNA isolation methods were various, according to the downstream applications. Protocols that included lysozyme produced a higher amount of DNA than the autoclave method. Lysozyme treatment combined with silica -guanidinethiocyanate procedure was the efficient protocol with affordable cost for routine lysis of L. acidophilus bacteria. Appropriate DNA concentration and quality were obtained through this method comparable to those of the commercial kit. Inversely, autoclave treatment had little effect on the breakage of the cell walls indicating low concentrations of extracted DNAs. This method could not completely break down all the bacterial cell walls. However, the breakage of low numbers of cell walls was microscopically observed in the supernatant of the autoclaved cell suspension. The quality of this protocol was found to be adequate for performing direct polymerase chain reaction (PCR) assay on samples with large amounts of lactobacilli. These conclusions suggest attentively selecting the DNA extraction method based on the planned downstream analysis of PCR products. [ABSTRACT FROM AUTHOR]

Plant pathogenic fungi cause severe yield losses and mycotoxin contamination in crops. The precise and rapid detection of fungal pathogens is essential for effective disease management. Sequencing universal DNA barcodes has become the standard method for the diagnosis of fungal diseases, as well as for identification and phylogenetic analysis. A major bottleneck in obtaining DNA sequence data from many samples was the laborious and time-consuming process of sample preparation for genomic DNA. Here, we describe a direct PCR approach that bypasses the DNA extraction steps to streamline the molecular identification of fungal species. Using a direct PCR approach, we successfully sequenced the nuclear ribosomal internal transcribed spacer (ITS) region for the representatives of major fungal lineages. To demonstrate the usefulness of this approach, we performed a phylogenetic analysis of the Fusarium fujikuroi species complex, which causes bakanae ("foolish seedling") disease of rice and mycotoxin contamination. A total of 28 candidate strains were isolated from rice seeds in the Republic of Korea, and the identity of the isolates was determined using the DNA sequence of both ITS and translation elongation factor 1-α regions. In addition, 17 F. fujikuroi isolates were examined for fumonisin (FB) production in rice medium using an enzyme-linked immunosorbent assay. Phylogenetic and toxigenic analyses showed that the F. fujikuroi strains could be distinguished into two groups: FB producers (B14-type) and non-producers (B20-type). These results will accelerate the molecular identification of fungal pathogens and facilitate the effective management of fungal diseases. [ABSTRACT FROM AUTHOR]

Macrophomina phaseolina (Tassi) Goid is the causal agent of charcoal rot and vine decline in cucurbits such as watermelon. Molecular methods have been used for rapid identification. However, a large number of steps used reduces its applicability. This study aimed to detect M. phaseolina in watermelon from producing areas in Northeastern Brazil by direct PCR. Plant tissue samples were collected from seven producing areas and the DNA was extracted using the CTAB method. Amplifications were performed by direct PCR using the MpKFI/MpKRI primers, then the PCR products were subjected to agarose gel electrophoresis and sequenced. Amplicons of 350 bp were observed in stem tissue samples from three areas. The identity of the samples was confirmed by sequencing. This study represents the first molecular diagnosis of M. phaseolina associated with watermelon in Northeastern Brazil. The methodology presented here can be applied for a reliable and simple diagnosis of the pathogen in other crops. [ABSTRACT FROM AUTHOR]

Accurate estimation of cell viability is crucial in various applications such as cytotoxicity testing and routine cell culture on both industrial and laboratory scales. For this, the real-time monitoring of cell status would be beneficial. Conventional cell-based assays for cell viability have limitations in sensitivity and time-effectiveness. Analysis of cell-free DNA (cfDNA) in (culture) media is a good alternative as cfDNA release are a well-known phenomenon during cell death. We demonstrate a direct digital PCR (dPCR) method to estimate cell viability by analyzing cfDNA in media during induced cell death. After validating the duplex dPCR method for short and long amplicons of the SMAD4 and RPP30 loci, we determined that a media volume of 2 μL is feasible to measure the target DNA copy number with minimal negative effects on amplification. dPCR inhibition was evident with a higher media volume per reaction targeting long amplicons. Next, we applied our dPCR method using media cfDNA and other conventional methods to the monitoring of camptothecin (CPT)-induced cell death. Copy numbers increased significantly after 4 h of CPT treatment, showing a fold change of approximately 4–6 compared to the controls. Cell-based assays such as the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay and annexin V/7-AAD assay also indicated increased cell death at 4 h, but the trypan blue exclusion assay did not. The developed media cfDNA direct dPCR method allows for efficient measurements of the degree of cell viability. Unlike other conventional cell-based assays, our method has advantages of no loss of cultured cells and the ability to implement online analysis. Accurate and sensitive media cfDNA analysis using dPCR can be adopted in various applications such as determining cytotoxicity levels in large-scale bioreactors or screening for effective anticancer drugs. [Display omitted] • A direct digital PCR method is developed to assess cell viability. • Duplex digital PCR assays are validated to directly measure cfDNA in media. • Amount of cfDNA increases during induced cell death, matching traditional assays. • The simple and sensitive method enables real-time viability testing without cell loss. [ABSTRACT FROM AUTHOR]

Macrofungi are valuable resources as novel drug candidates, new biomaterials, and edible materials. Recently, genetic approaches pertaining to macrofungi have been continuously growing for their identification, molecular breeding, and genetic engineering. However, purification and amplification of fungal DNA is challenging because of the rigid cell wall and presence of PCR inhibitory metabolites. Here, we established a direct PCR method to provide a rapid and efficient method for PCR-grade macrofungal DNA preparation applicable to both conventional PCR and real-time PCR. We first optimized the procedure of lysis and PCR using the mycelia of Lentinula edodes, one of the most widely consumed macrofungal species. Lysates prepared by neutralizing with (NH4)2SO4 after heating the mycelia in a mixture of TE buffer and KOH at 65℃ for 10 min showed successful amplification in both conventional and real-time PCR. Moreover, the addition of bovine serum albumin to the PCR mixture enhanced the amplification in conventional PCR. Using this method, we successfully amplified not only internal transcribed spacer fragments but also low-copy genes ranging in length from 500 to 3,000 bp. Next, we applied this method to 62 different species (54 genera) of macrofungi, including edible mushrooms, such as Pleurotus ostreatus, and medicinal mushrooms such as Cordyceps militaris. It was found that our method is widely applicable to both ascomycetes and basidiomycetes. We expect that our method will contribute to accelerating PCR-based approaches, such as molecular identification, DNA marker typing, gene cloning, and transformant screening, in macrofungal studies.

Background: Diagnosis with reverse transcriptase polymerase chain reaction (RT-PCR) test is a very important step for the control of the COVID-19 pandemic. The aim of this study is to compare the RT-PCR results of the samples taken directly from the viral transport medium (VTM) without extraction with the RT-PCR results of two different extraction methods, one automated and the other manual, in the diagnosis of COVID-19. Methods: Among the respiratory tract samples sent to Sakarya Training and Research Hospital Microbiology Laboratory for COVID-PCR study, 20 negative and 43 positive samples with different cycle threshold (CT) values were included in the study. Both manual nucleic acid isolation with the vNAT isolation kit (Bioeksen, Turkey) and automatic nucleic acid isolation with the EZ1 Virus Mini Kit v2.0 in the isolation device were performed simultaneously from the patient samples included in the study and the results were compared. Results: The mean Ct values of the samples were found to be 21.58 using manual vNAT as the extraction method, 17.63 using the automated magnetic bead method, and 21.45 in PCR from direct VTM without extraction. When the automatic magnetic beads extraction method was taken as the reference method, the sensitivity of direct PCR was 97.3%, the specificity was 95%, the positive predictive value was 97.3%, and the negative predictive value was 95%. Phi coefficients were found to be 0.927 between vNAT and direct PCR, 1 between vNAT and EZ1, and 0.922 between direct PCR and EZ1. Conclusions: Direct PCR has advantages such as eliminating RNA extraction and purification steps, providing a shorter detection time, and using less labor and less consumables without reducing the diagnostic accuracy. It is thought that this method can help as a useful process management for the control of the epidemic in countries with limited resources. [ABSTRACT FROM AUTHOR]

Bloodstains on fabrics may be washed or cleaned to eliminate incriminating evidence. These actions reduce the chances of obtaining an interpretable DNA profile. Previous studies have shown that conventional short-tandem repeat (STR) typing is affected by various factors associated with washing or laundering of stains. Here, we aim to increase the chances of obtaining interpretable STR profiles from laundered bloodstains using direct PCR. Preliminary investigations showed direct STR typing resulted in more alleles compared to conventional STR typing. We then further investigated the following factors with direct STR typing: fabric type (cotton, polyester, and denim), washing method (hand-washing and machine-washing), type of detergents (powder and liquid), washing temperature (cold to 90 °C), pretreatment agents (sodium hypochlorite and hydrogen peroxide), and the number of washes (one, three, and five). Direct PCR could be successfully used for STR typing from laundered bloodstains with very high success rates. Among the three fabric types, only denim negatively affected direct STR typing, while laundering of bloodstains on cotton and polyester had a negligible effect as mostly full profiles were obtained. Multiple washes resulted in a decrease in both the numbers of alleles and peak heights. Surprisingly, washing method, type of detergent, washing temperature, and pretreatment agents only had minimal to no effect on STR profile quality. Due to the robustness and sensitivity of direct STR typing from laundered bloodstains, the method could be beneficial for violent crime investigations in forensic DNA laboratories worldwide. [ABSTRACT FROM AUTHOR]

Macrophomina phaseolina (Tassi) Goid is the causal agent of charcoal rot and vine decline in cucurbits such as watermelon. Molecular methods have been used for rapid identification. However, a large number of steps used reduces its applicability. This study aimed to detect M. phaseolina in watermelon from producing areas in Northeastern Brazil by direct PCR. Plant tissue samples were collected from seven producing areas and the DNA was extracted using the CTAB method. Amplifications were performed by direct PCR using the MpKFI/MpKRI primers, then the PCR products were subjected to agarose gel electrophoresis and sequenced. Amplicons of 350 bp were observed in stem tissue samples from three areas. The identity of the samples was confirmed by sequencing. This study represents the first molecular diagnosis of M. phaseolina associated with watermelon in Northeastern Brazil. The methodology presented here can be applied for a reliable and simple diagnosis of the pathogen in other crops.

Taq DNA polymerases have played an important role in molecular biology for several years and are frequently used for polymerase chain reaction (PCR); hence, there is an increasing interest in developing a convenient method for preparing Taq DNA polymerase for routine use in laboratories. We developed a method using Escherichia coli (E. coli) that expresses thermostable Taq DNA polymerase directly in the PCR without purification. The Taq gene was transformed into E. coli and expressed. After overnight incubation and washing, E. coli-expressing Taq DNA polymerase (EcoliTaq) was used as the DNA polymerase without purification. EcoliTaq showed activity comparable to that of commercial DNA polymerase and remained stable for 3 months. With a high-pH buffer containing 2% Tween 20 and 0.4 M trehalose, EcoliTaq facilitated direct PCR amplification from anticoagulated whole blood samples. EcoliTaq exhibited good performance in allele-specific PCR using both purified DNA and whole blood samples. Furthermore, it proved to be useful as a DNA polymerase in hot-start PCR by effectively minimizing non-specific amplification. We developed a simple and cost-effective direct and hot-start PCR method in which EcoliTaq was used directly as a PCR enzyme, thus eliminating the laborious and time-consuming steps of polymerase purification.

Molecular diagnostic assays for cholera detection have superior sensitivity to conventional assays and are now being accepted as the new standard method, especially the real-time PCR/RT-PCR. However, limited throughput capacity and long detection duration prevent them from detecting more specimens and more targets in one turnaround time simultaneously. In this study, we utilized nucleic acid extraction-free, direct RT-PCR and high-speed amplification to develop a novel multiplex assay, a quadplex direct one-tube real-time RT-PCR assay, for rapid detection of the serogroup and cholera toxin toxigenicity of Vibrio cholerae targeting the epsM, ctxA, rfb-O1, and rfb-O139 genes. Performance of the multiplex assay was evaluated by comparison with the monoplex real-time PCR assay according to the China Cholera Prevention Manual. Detection data from clinical specimens showed that the new assay had good diagnostic sensitivities for epsM (100%, n = 301), ctxA (100%, n = 125), rfb-O1 (100%, n = 85), and rfb-O139 (97.87%, n = 49). Analysis of the analytical sensitivities with serial dilutions of positive standards showed that the detection limits of the new assay for Vibrio cholerae epsM,ctxA,rfb-O1, and rfb-O139 were up to 200, 590, 115, and 1052 copies per mL lower than the monoplex real-time PCR (910, 345, and 1616 copies/mL respectively, for ctxA,rfb-O1, and rfb-O139). The results indicate that the multiplex assay is a rapid, sensitive, specific, and easy-to-use detection tool for Vibrio cholerae, especially suitable for rapid identification and screening detection of mass specimens. [ABSTRACT FROM AUTHOR]

Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction. Furthermore, by the CRISPR/Cas12a, the biosensor addressed false positives caused by non-specific amplification and had high sensitivity with the actual limit of detection (LOD) of 7.6×10−4 ng·μL−1 (4 copies·μL−1). In addition, the strategy was built on the lateral flow assay (LFA) strip to achieve visual and portable detection for point-of-care testing. Moreover, the biosensor by a fluorometer and LFA strip showed a high accuracy to rival qPCR in actual sample detection. Therefore, the biosensor is an ultra-sensitive and specific tool that can replace traditional methods. Key points: • No nucleic acid extraction, direct PCR-simplified steps, and reduced time and cost • CRISPR/Cas12a solved the false positives caused by nonspecific amplification • The combination of the LFA strip and biosensor is more convenient for POC detection [ABSTRACT FROM AUTHOR]

Recovery of touch DNA from fired ammunition casings may provide vital forensic evidence for investigation and/or prosecution of firearm offences. Previous studies employing a direct PCR approach, where the traditional DNA extraction process is bypassed, have demonstrated an improved profiling success rate from some types of ammunition. To assess the potential value of these techniques to recover touch DNA from fired shotgun ammunition, direct PCR was evaluated for its ability to recover DNA profiles from fired 12 gauge Buckshot OOSG shotgun cartridges as compared to routine swabbing with DNA extraction. In this study, swabs subjected to direct PCR gave a significantly lower recovery of alleles than those undergoing DNA extraction. The presence of PCR inhibitors that were produced or deposited during the firing process may be the cause of these poorer results. The success of a direct PCR approach may therefore be directly related to the type of ammunition or firearm used and should therefore be specifically tested for individual case scenarios prior to employing it in forensic casework. [ABSTRACT FROM AUTHOR]

Testing for potato viruses is globally very important to prevent a critical shortage of potato supply. In most countries, testing is obligated by law. In Germany, seed potatoes are monitored for six viruses: PLRV, PVY, PVM, PVA, PVX and PVS. They can cause up to 90% loss of potato tubers in the field. Common methods currently used for testing are ELISA and conventional real-time PCR, but both are very time-consuming, and the former needs a high capacity of green houses and human resources, the latter elaborate RNA extraction steps. Recently, we proposed a new method called real-time DiRT-PCR which enables us to test for PLRV, PVY and PVS along with an internal control in three duplex real-time PCR reactions directly on diluted tuber sap. In this study, we describe the first TaqMan® assay for PVM published so far and embed it into a multiplex system to detect the remaining viruses. We are now able to sensitively test for the presence of six viruses in two multiplex reactions using the real-time DiRT-PCR without RNA purification. [ABSTRACT FROM AUTHOR]

Abstract Brucellosis is a zoonotic disease that cause a significant economic losses for cattle industries worldwide. A rapid, precise and accurate diagnosis technique for diagnosis of brucellosis in all stages of the infection is definitely required. Blood-samples are widely used for PCR-based DNA analysis because they are easily collected, handled, and processed. Direct PCR analysis without DNA extraction has been attempted to reduce time and costs for routine analysis. This approach is promising but is still limited by the presence of PCR inhibitors that is naturally found in the blood samples. The objective of this study was to compare the effectivity of direct PCR technique with or without DNA extraction for detection of Brucella abortus in the blood samples. Three whole-blood samples from brucella infected dairy cattle and five whole-blood samples from beef cattle that having abortion were used as samples in this study. A pair of bcsp31 primers and IS711 primers were used for amplification of genus-specific and species-specific of Brucella. The results showed that amplicon in the position of 223 bp and 498 bp that are specific for B. abortus were detected from all of the samples that were analyzed on 1.5% agarose gels. Based on the result it could be concluded that direct PCR analyses without DNA extraction is a sensitive, specific, simple, rapid and inexpensive assay for detecting B. abortus in the whole blood samples for either dairy or beef cattle and therefore it could improve the existing surveillance and control programs for brucellosis. Keywords : brucellosis; direct PCR; PCR inhibitor; whole-blood sample; without DNA extraction Abstrak Brucellosis adalah penyakit zoonosis yang menyebabkan kerugian ekonomi yang signifikan bagi industri ternak di seluruh dunia. Teknik diagnosis yang cepat, tepat dan akurat yang dapat digunakan untuk diagnosis brucellosis pada semua tahap infeksi sangat diperlukan. Sampel darah banyak digunakan untuk analisis PCR berbasis DNA karena mudah untuk dikoleksi, ditangani, dan diproses. Metoda PCR langsung tanpa didahului dengan ekstraksi DNA dikembangkan dengan tujuan penghematan waktu dan beaya untuk analisa secara rutin. Tehnik ini sangat menjanjikan tetapi memiliki keterbatasan karena adanya senyawa penghambat PCR yang secara alami terkandung di dalam sampel darah . Tujuan dari penelitian ini adalah membandingkan efektifitas antara uji PCR secara langsung dengan ekstraksi dan tanpa ekstraksi DNA untuk deteksi Brucella abortus di dalam darah. Tiga ( 3 ) sampel darah-EDTA yang berasal dari sapi penderita brucellosis dan 5 sampel darah-EDTA dari sapi potong yang mengalami abortus digunakan sebagai sampel dalam penelitian ini. Pasangan primer bcsp31 dan primer IS711 untuk amplifikasi gen dan species specific digunakan dalam penelitian. Hasil menunjukkan bahwa amplikon/pita pada posisi 223 bp dan 498 bp yang spesifik untuk Brucella abortus terdeteksi dari semua sampel yang dianalisa dengan gel agarosa 1,5%. Berdasarkan hasil penelitian dapat disimpulkan bahwa uji PCR secara langsung tanpa didahului dengan ekstraksi DNA merupakan tehnik yang sensitif, spesifik, sederhana, cepat dan murah untuk deteksi B. abortus di dalam sampel darah baik sapi perah maupun sapi potong dan oleh karena itu diharapkan dapat digunakan untuk memperbaiki program kontrol dan survailance yang telah ada untuk brucellosis. Kata kunci : brucellosis; PCR langsung; penghambat PCR; sampel darah-utuh; tanpa ekstraksi DNA

Genetic engineering is one of the strategies for developing nitrogen (N)-use-efficient rice (Oryza sativa) varieties. One gene that plays an indirect role in N metabolism is alanine aminotransferase (AlaAT). It can efficiently increase N content and crop yield. In a previous study, the tomato AlaAT gene (LeAlaAT) was successfully isolated and introduced into ‘Mekongga’ rice. The present research was conducted during 2018 and 2019 at the Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD), Bogor, Indonesia. The objectives of the present study were to perform the molecular characterization of LeAlaAT ‘Mekongga’ rice lines on the basis of the hpt marker gene, the direct PCR of the LeAlaAT fragment, and the phenotypic evaluation of the selected LeAlaAT T1 ‘Mekongga’ rice lines in response to different N fertilizer rates (0 kg ha−1 [control] and 60, 90, and 120 kg ha−1). This research involved three activities, namely (1) Southern blot analysis, (2) direct PCR, and (3) N use efficiency (NUE) test of ‘Mekongga’ transgenic lines. Southern blot analysis revealed that in T0 transgenic lines, the copy number of the hpt marker gene varied from 1 to 3. Direct PCR confirmed the presence of the AlaAT fragment in the T1 generation of five ‘Mekongga’ transgenic lines. The five transgenic lines showed high panicle number, biomass weight, shoot dry weight, and total grain weight under 120 kg ha−1 nitrogen. The high agronomical NUE of transgenic lines under 120 kg ha−1 N implied that the transgenic rice lines have the potential for efficient N use at a certain minimum level of N (120 kg ha−1 of nitrogen) and should be further evaluated at high N levels. [ABSTRACT FROM AUTHOR]

In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples.

Hemoglobin Constant Spring (Hb CS) and Hb Pakse' (PS) are the common non-deletional α+-thalassemia found in Thailand. These two variants can cause severe thalassemia syndromes, especially in fetus and neonate. Molecular diagnosis is the only confirmatory method because Hb CS and Hb PS are usually missed by routine screening and Hb analysis. Therefore, we aimed to develop rapid direct PCR for the diagnosis of Hb CS and PS genes. Multiplex direct PCR assays for identifying the Hb CS and PS genes in whole blood (WB) and amniotic fluid (AF) specimens were developed. The assays were firstly validated on 290 unrelated whole blood specimens. Hb CS and PS carriers were identified in 67 (23.1%) and 6 (2.1%) cases, respectively. A 100% concordant result as compared to routine PCR assay was observed. The direct PCR assays have been applied successfully for prenatal diagnosis in two families. The result showed that the fetuses were affected by homozygous Hb CS and compound heterozygous Hb CS/Hb PS. Accurate prenatal diagnosis of these families was observed using the newly developed assays. These assays should be applicable in routine thalassemia diagnostics as well as in the large-scale screening of Hb CS and PS in the region. [ABSTRACT FROM AUTHOR]

With an increase in the application of direct PCR to items of forensic relevance, as well as the array of STR kits available for amplification, the need for a comprehensive investigation into the optimum STR panel for this workflow has arisen. Here we examine the relative STR amplification success of touch DNA on a range of substrates, with surface properties typical of those found in forensic investigation, using six commercially available STR kits: GlobalFiler®, Identifiler® Plus, Identifiler® Direct, VeriFiler™ Plus, Investigator® 24Plex QS, and PowerPlex® 21. We report on the percentage of possible donor alleles amplified per profile, and the number of samples that resulted in informative genetic data (≥12 autosomal alleles). We also include comment on the ease of interpretation of the resulting electropherograms for each of these six STR kits, when applied within a direct PCR workflow. Donors of known shedder status deposited DNA by handling one of five substrates (glass slide, matchstick, insulated wire, circuit board, and a plastic ziplock bag) for 15 s, 15 min post-handwashing with water. Each item was touched in triplicate by each volunteer for amplification with each STR kit resulting in a dataset of 720 samples. The difference in the number of profiles considered to be informative was found to be statistically significant when comparing STR kits (p = 0.0011) and donors (p = 2e-07), but not when considering substrates (p = 0.15). Identifiler® Plus amplification resulted in the highest profile coverage and second highest percentage of informative profiles. VeriFiler™ Plus generated informative profiles in the largest number of samples (94%) with PowerPlex® 21 amplification resulting in the fewest (79%). There was no significant difference between Investigator® 24Plex QS and GlobalFiler® in any empirical consideration; however, baseline noise and artefact presence made Investigator 24PlexQS profiles more difficult to analyse.

Insectivorous bats may significantly contribute to human well-being by suppressing pest insects and possibly preventing the emergence of diseases. To understand the roles these bats play in their ecosystems, a diet analysis of their guano can be carried out. However, each diet analysis method has its drawbacks, e.g., some soft-bodied insects might be missed in microscopic analysis of guano. We aimed to examine the diet of the wrinkle-lipped free-tailed bats (Chaerephon plicatus) using direct PCR and DGGE (denaturing gradient gel electrophoresis) technique. Sequencing was done for 240 guano samples collected from two caves in Thailand over the course of a year. Direct PCR and DGGE was successfully applied for bat guano analysis. Seventy-six Operational Taxonomic Units were identified, in which 25 were determined to the species level. Diptera was the most abundant insect order found in bats' diet, with a percentage frequency of occurrence (%FOO) of 32.8%, followed by Hemiptera (27.2%), Lepidoptera (24.1%) and Coleoptera (10.3%). Hemipterans were preferred during active rice-growing months, while dipterans were consumed year-round. Eight known crop pests were found, and the brown planthopper (Nilaparvata lugens) was the most dominant throughout the year. Mosquitoes also substantially contributed to the bat's diet. The bats probably encounter these insects during insect dispersal at high elevations. The prey species recorded strongly indicated that this bat plays a role in facilitating rice and crop productions, which increases food security. In addition, this bat may play a role in suppressing potential disease-carrying insects such as various species of mosquitoes. We urge local and international authorities to increase conservation efforts and that similar studies should be done with other bat species. [ABSTRACT FROM AUTHOR]

Engineered plants have been widely produced for fundamental and practical use. Several methods have been developed for genetically modified crop detection and quantification; however; they still laborious and expensive. Efforts are needed to set-up diagnosis-oriented techniques as alternatives to overcome DNA extraction which remains a tedious and time-consuming procedure. Here, we established a standard direct PCR workflow using a regular Taq polymerase without prior DNA purification over a wide range of plant species. Only a small amount of fresh tissue allowed direct amplification of target gene sequences. Evaluation of accuracy, sensitivity, and reproducibility of direct PCR assay was investigated for proof-of-concept, and subsequently applied to gene detection assays and rapid transgenic revealing. The newly established method achieved full success and has amplified constitutive housekeeping genes from several plant specimens in a reproducible manner with high-quality sequencing profiles. In our case, the screening of transgenic plants confirmed that both the gfp-ER reporter gene and the npt II selectable marker were integrated into the plant genome. This direct PCR approach provides a powerful tool for large-scale PCR-based gene detection making DNA purification irrelevant. It could be easily implemented for downstream applications in the field of genetic fingerprinting, plant biotechnology, and functional genomics. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) have been intensively investigated and widely applied in regenerative medicine and immune modulation. However, their efficacy declines during the aging or disease process. Thus, genome-edited MSCs with over-expression or inhibition of specific genes hold a great deal of promise in terms of their therapeutic application. Here we optimized the direct PCR approach for rapid identification of genome-edited MSCs with only ten cells required, which reduces the time and labor to expand the MSC colonies. Combined with our previously optimized guide RNA structure and plasmid construction strategy for Cas9, we successfully identified MSC colonies over-expressing IL-10 in the AAVS1 locus.

Molecular diagnostic assays for cholera detection have superior sensitivity to conventional assays and are now being accepted as the new standard method, especially the real-time PCR/RT-PCR. However, limited throughput capacity and long detection duration prevent them from detecting more specimens and more targets in one turnaround time simultaneously. In this study, we utilized nucleic acid extraction-free, direct RT-PCR and high-speed amplification to develop a novel multiplex assay, a quadplex direct one-tube real-time RT-PCR assay, for rapid detection of the serogroup and cholera toxin toxigenicity of Vibrio cholerae targeting the epsM, ctxA, rfb-O1, and rfb-O139 genes. Performance of the multiplex assay was evaluated by comparison with the monoplex real-time PCR assay according to the China Cholera Prevention Manual. Detection data from clinical specimens showed that the new assay had good diagnostic sensitivities for epsM (100%, n = 301), ctxA (100%, n = 125), rfb-O1 (100%, n = 85), and rfb-O139 (97.87%, n = 49). Analysis of the analytical sensitivities with serial dilutions of positive standards showed that the detection limits of the new assay for Vibrio cholerae epsM,ctxA,rfb-O1, and rfb-O139 were up to 200, 590, 115, and 1052 copies per mL lower than the monoplex real-time PCR (910, 345, and 1616 copies/mL respectively, for ctxA,rfb-O1, and rfb-O139). The results indicate that the multiplex assay is a rapid, sensitive, specific, and easy-to-use detection tool for Vibrio cholerae, especially suitable for rapid identification and screening detection of mass specimens.

The extraction of DNA is a fairly standard procedure routinely undertaken in biological academic and research settings. In a forensic DNA context, DNA extraction is the crucial first step toward the generation of DNA profiles used to support convictions or exonerations in the criminal justice system. However, it is often complicated by the additional challenge of yielding sufficient quantities of clean DNA that is free from environmental inhibitors, from a wide spectrum of sample types which may contain limited quantities of biological material. While improvements in methods and technology over the decades have sped up and simplified the process of DNA extraction, several issues remain to be fully addressed—such as the incomplete separation of spermic DNA from sperm‐epithelial cell mixtures, increasing demands for ever‐faster DNA results, or even on‐scene DNA processing, and processing of difficult samples such as skeletal remains or samples contaminated by Chemical, Biological, or Radiological (CBR) agents. These challenges are often compounded by high work volumes and limited quantities of starting material. As such, this review focuses on recent trends and developments in four areas of forensic DNA extraction: (a) analysis of sexual assault evidence which often represents a significant portion of a forensic DNA laboratory's casework, (b) use of portable rapid DNA instruments which has gained much traction among law enforcement in recent times, (c) DNA extraction from difficult and CBR‐contaminated samples which, albeit rare, would be essential in a DNA laboratory's toolbox in the event of an incident, and (d) bypassing DNA extraction altogether. This article is categorized under:Forensic Biology > Forensic DNA Technologies [ABSTRACT FROM AUTHOR]

Featured Application: This paper's application of the proposed novel method is reducing the ski-slope effect in direct PCR, thus aiding more efficient amplification of DNA obtained from crime scenes. Analytical techniques such as DNA profiling are widely used in various fields, including forensic science, and novel technologies such as direct polymerase chain reaction (PCR) amplification are continuously being developed in order to acquire DNA profiles efficiently. However, non-specific amplification may occur depending on the quality of the crime scene evidence and amplification methods employed. In particular, the ski-slope effect observed in direct PCR amplification has led to inaccurate interpretations of the DNA profile results. In this study, we aimed to reduce the ski-slope effect by using dimethyl sulfoxide (DMSO) in direct PCR. We confirmed that DMSO (3.75%, v/v) increased the amplification yield of large-sized DNA sequences more than that of small-sized ones. Using 50 Korean buccal samples, we further demonstrated that DMSO reduced the ski-slope effect in direct PCR. These results suggest that the experimental method developed in this study is suitable for direct PCR and may help to successfully obtain DNA profiles from various types of evidence at crime scenes. [ABSTRACT FROM AUTHOR]

CRISPR/Cas systems (Clustered regularly interspaced palindromic repeats / CRISPR-associated) are rapidly becoming a commonplace and popular tool for gene editing in research and clinical contexts. However, the quality of CRISPR/Cas experiments depends heavily on the guide RNA (gRNA) design; therefore, a reliable, easy, and rapid method for verifying gRNA cleavage efficacy is necessary. Engineered nuclease-induced translocations (ENIT) are an easy and cost-efficient method for the verification of gRNA efficacy, which involves tracking induced chromosomal mutations, using polymerase chain reaction (PCR). We have customized this method using both direct PCR and nested PCR approaches and have been able to reduce the sample preparation time. We present a simple and reliable gRNA testing approach that requires no specific enzymes or equipment. • The approach requires only routinely used enzymes and equipment. • Cost- and time-efficient, requiring approximately 30 min for PCR sample preparation, without requiring DNA purification. • High sensitivity, with induced translocation detected in 100 of 10,000 cells in the general population.

Herbarium collections provide an essential basis for a wide array of biological research and, with development of DNA-based methods, they have become an invaluable material for genetic analyses. Yet, the use of such material is hindered by technical limitations related to DNA degradation and to quantity of biological material. The latter is inherent for some biological groups, as best exemplified by myxomycetes which form minute sporophores. It is estimated that ca. two-thirds of myxomycete taxa are represented by extremely scanty material. As DNA isolation methods applied so far in myxomycete studies require destructive sampling of many sporophores, a large part of described diversity of the group remains unavailable for phylogenetic studies or barcoding. Here, we tested several procedures of DNA isolation and amplification to seek for an efficient and possibly non-destructive method of sampling. Tests were based on herbarium specimens of 19 species representing different taxonomic orders. We assayed several variants of isolation based on silica gel membrane columns, and a newly designed procedure using highly reduced amount of biological material (small portion of spores), based on fine disruption of spores and direct PCR. While the most frequently used column-based method led to PCR success in 89.5% of samples when a large amount of material was used, its performance dropped to 52% when based on single sporophores. Single sporophores provided amplicons in 89.5% of samples when using a kit dedicated to low-amount DNA samples. Our new procedure appeared the most effective (94.7%) while it used only a small fraction of spores, being nearly non-destructive; it was also the most cost-effective. We thus demonstrate that combination of adequate handling of spore micro-disruption coupled with application of direct PCR can be an efficient way to circumvent technical limitations for genetic studies in myxomycetes and thus can substantially improve taxon sampling for phylogeny and barcoding. Additionally, this approach gives a unique possibility to apply both molecular and morphological assays to the same structure (sporophore), which then can be further stored as documentation.

Infectious diseases are considered the greatest threat to the modern high-density shrimp aquaculture industry. Specificity, rapidity, and sensitivity of molecular diagnostic methods for the detection of asymptomatic infected shrimp allows preventive measures to be taken before disease outbreaks. Routine molecular detection of pathogens in infected shrimp can be made easier with the use of a direct polymerase chain reaction (PCR). In this study, four direct PCR reagent brands were tested, and results showed that the detection signal of direct PCR in hepatopancreatic tissue was more severely affected. In addition, portable capillary electrophoresis was applied to improve sensitivity and specificity, resulting in a pathogen detection limit of 25 copies/PCR-reaction. Juvenile shrimp from five different aquaculture ponds were tested for white spot syndrome virus infection, and the results were consistent with the Organization for Animal Health’s certified standard method. Furthermore, this methodology could be used to examine single post larvae shrimp. The overall detection time was reduced by more than 58.2%. Therefore, the combination of direct PCR and capillary electrophoresis for on-site examination is valuable and has potential as a suitable tool for diagnostic, epidemiological, and pathological studies of shrimp aquaculture.

Direct polymerase chain reaction method was use to amplify a thaumatin-like sequence of Vitis vinifera L. in grapes as well as in stormy wines and musts. Thaumatin-like proteins (TLPs) of Vitis vinifera possess beside its function in abiotic and biotic stress response another one - they are able to cause protein haze in wine unless removed prior to bottling. Direct PCR is an approach where omission of DNA extraction is typical prior the amplification of the target site of plant genome. Crude extract or small pieces of plant tissues are used in the analysis directly without steps of extraction and purification of gDNA. The biological material that was used in analysis was collected during August - October 2017 in local stores and winery Sabo and comprises from cultivars Iršai, Muškát, Savignon Blanc, Svätovavrinecké, Dornfelder and Pálava. Direct PCR was performed by a cutted piece of grape tissue and a dilution buffer was use in 1:2 for stormy wine or must, respectively. Direct amplification of thaumatin-like protein sequence of Vitis vinifera was performed along with the control reactions with the primers for conserved region of plant chloroplast. Possitive amplification of thaumatin-like allergen sequence resulted in 570 bp amplicon. The most abundant amplicons were amplified in stormy wines, followed by musts and the amplicons from grapes were weaker when comparing them to others. The amplicon specificity checking of obtained PCR product of thaumatin-like allergen was performed by restriction cleavage by Psi I and resulted in restriction amplicons of the 80 bp, 81 bp, 94 bp and 315 bp in length. Confirmation of the amplicon specificity by restriction cleavage support the potential of direct PCR to become a reproducible method that will be fully applicable in routine analysis of not only plant genomes in the future, but it was demonstrated, that it works in liquids, too.

• For the first time, direct PCR of buccal lysates was explored in a post-mortem setting. • 79% of samples yielded full DNA profiles, and failure was attributed to low DNA yield. • A full DNA profile was obtained from a decedent who was stored for 887 days. • DNA profile success was not associated with duration of body storage. Forensic DNA profiling is a standard method used in the attempt to identify deceased individuals. In routine investigations, and if available, the preferred sample type is usually blood. However, this requires the invasive re-opening of the body, days or weeks after the autopsy, which is undesirable in resource-constrained mortuary settings. Motivated by the ease of sampling as well as reduced health and safety risks, this study aimed to establish the success rate of generating a full DNA profile on first attempt from buccal swab lysates using a direct PCR approach. Buccal swab samples were collected from 100 unidentified deceased males, and were subjected to direct DNA profiling with use of the Promega PowerPlex® Y23 Kit. At the time of sample collection, these individuals had been stored for between 1 and 887 days. This study shows that full DNA profiles were initially obtained from 73% of samples, which constitutes the first empirical data pertaining to first time success rates of direct PCR from post-mortem buccal lysates. Further investigation of partial and failed DNA profiles using real-time PCR showed that samples did not contain PCR inhibitors, DNA was not degraded, but DNA concentration was particularly low. Repeating DNA profiling with increased lysate input and extra PCR cycles yielded an additional six full DNA profiles, resulting in an overall success rate of 79%. Overall, DNA profile success rate was not associated with the duration of storage (p = 0.387). Lastly, massively parallel sequencing with the ForenSeq™ Signature DNA Prep kit provided more informative profiles for three additional samples. These results indicate that blood should therefore remain the sample of choice in a post-mortem setting, yet buccal lysates hold potential to be optimised further, which may ease the human identification workflow. [ABSTRACT FROM AUTHOR]

• To-date review of direct PCR in forensic science. • Direct PCR in forensic science is increasing due to the sensitivity it offers. • Success in obtaining DNA profiles from challenging samples is found with direct PCR. • Challenges in incorporating this technique into forensic workflow are discussed. [ABSTRACT FROM AUTHOR]

Variation has been reported in the amount of DNA accumulating on the skin of individuals. A shedder status is the propensity of a person to transfer DNA to a substrate by touch. In previous tests of shedders, individuals washed their hands and after 15 minutes made contact with substrates at time points up to 180 minutes after handwashing. No examination has looked at the accumulation of cellular material between time zero and this 15 minute time point. Here we present the results of an examination of cellular material within thumbprints at time points 0, 2, 5, 15 and 60 minutes after handwashing using donors who are representative of heavy, intermediate and light shedders. The rate of accumulation of cellular material and the total amount detected in thumbprints showed a difference between these donors, but for all donors the initial rate of accumulation is surprisingly fast. Informative STR profiles can be generated only 2 minutes after handwashing from 100%, 33% and none of the heavy, intermediate and light shedders, respectively. These results confirmed that there was a correlation between the cellular material present on the thumbprint and the percentage success of an STR profile for each individual and time point. [ABSTRACT FROM AUTHOR]

The light brown apple moth (LBAM) was first detected in Alameda County, California, USA, in fall of 2006, triggering a major government response aimed at containment and eradication. The augmentative release of resident egg parasitoids was highlighted among several control methods to prevent the spread of LBAM in California. Egg parasitoids in the genus Trichogramma are utilised worldwide for the control of lepidopteran pests, including the control of LBAM in Australia. Surveys of the resident egg parasitoids attacking LBAM in California, carried out in 2009, resulted in the identification of five Trichogramma species: T. fasciatum, T. platneri, T. deion, T. nr pretiosum, and T. fuentesi. Of these, only T. platneri had previously been recorded in our study area. These initial identifications were based on ribosomal DNA sequences. To facilitate further monitoring of the distribution and abundance of these species, a species-specific multiplex-PCR (ssmPCR) was developed to allow rapid and affordable identification. To save further time and expense, we took away the traditional DNA extraction step and demonstrated that the method performs well when an intact wasp is placed directly into the amplification reaction. In all, per specimen identification is estimated to cost significantly less than one US dollar. [ABSTRACT FROM AUTHOR]

Molecular diagnostic methods have been used to supplement morphological methods in taxonomy. In this study, we used molecular methods to diagnose populations of dileptid ciliates, which commonly occur in terrestrial and semi-terrestrial habitats. Molecular investigations based on the comparison of DNA sequences have been carried out with a limited number of ciliate species due to insufficient genomic DNA for PCR because of their small population size and difficulties to maintain monocultures. The present study shows that there is a loss of DNA with conventional methods and proposes a novel approach: cells (stored at -20°C or fresh) are directly subjected to PCR, without being processed by any chemical treatment. The small subunit ribosomal DNA (SSU rDNA) (18S rRNA gene) of dileptid ciliates was successfully amplified by direct PCR, without DNA extraction, for single-cell and multi-cell samples. This method has been found to be simpler and especially useful for species that are rare and have small population sizes. [ABSTRACT FROM AUTHOR]

• Metal surfaces are difficult substrates for trace DNA recovery and amplification. • Metal cations interact with DNA via complex intercalation, irreversible covalent binding and groove association. • Five methods of touch DNA sampling include swabbing, tape lifting, soaking, Bardole M-VAC and direct PCR. • There is at most 26% DNA recovery success rate from cartridges, bullets and casings (CBCs). • The surface characteristics of metal substrates are relevant to nucleic acid persistence and recovery. Trace evidence such as touch (also known as contact) DNA has probative value as a vital forensic investigative tool that can lead to the identification and apprehension of a criminal. While the volume of touch DNA evidence items submitted to forensic laboratories has significantly increased, recovery and amplification of DNA from these items, especially from metal surfaces, remains challenging. Currently little is understood with regards to the underlying mechanisms of metal-DNA interactions in the context of forensic science and how this may impact on DNA recovery. An increased understanding of these mechanisms would allow optimisation of methods to improve outcomes when sampling these materials. This paper reviews the basis of DNA binding to metal substrates, the merits and limitations of current methods and future perspectives of improving recovery and amplification of touch DNA from metal surfaces of forensic interest. [ABSTRACT FROM AUTHOR]