Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

Establishment of rapid detection method for zika virus based on direct amplification RT-PCR technique / 国际检验医学杂志

Objective To establish a rapid detection method for zika virus based on direct amplification re-al-time fluorescent quantitative reverse transcription polymerase chain reaction(RT-PCR)technique.Methods A direct amplification RT-PCR technique for the rapid detection of zika virus in 5 samples(whole blood,serum,saliva,throat swab and urine)was established by using a special function DNA polymerase and a preferred PCR enhancer.Results The detection limits of the 5 samples were 103 PFU/mL in serum,102 PFU/mL in urine,throat swab,and saliva,and 104 PFU/mL in whole blood.The coefficient of goodness-fit of stand-ard curves was above 0.98,and the amplification efficiency was 90％-110％.Zika virus nucleic acid was suc-cessfully amplified,but non-zika virus nucleic acid was not amplified.Based on the repeatable detection of sam-ples from urine,whole blood,and saliva,the variation coefficient of 6 repeated Ct values at 106 PFU/mL and 102 PFU/mL concentrations were all＜5％.The zika virus detection method established by the direct amplifi-cation RT-PCR technique was consistent with the detection results of conventional RT-PCR technique.Only two serum samples were detected in eight zika virus samples,and the remaining 62 non-zika virus samples and 12 negative samples were not amplified.Conclusion A rapid detection method for zika virus based on direct ampli-fication RT-PCR technique is successfully established.The method is simple,rapid,sensitive and specific.

Effect of graphene oxide nanoparticles on in vitro growth of Fragaria x ananassa (Cameron Highlands white Strawberry) and evaluation of genetic stability using DAMD and ISSR markers.

Graphene oxide (GO) is a novel nanomaterial with distinct physical properties and significant biological applications. The use of GO in plant tissue culture offers several new properties and potential applications. This research is vital due to the growing need for innovative techniques to promote plant growth, improve plant productivity and mitigate challenges posed by environmental stressors. This study focused on the rare Cameron Highlands white strawberry plants (Fragaria x ananassa) and addressed issues such as callus production during direct shoot induction and hyperhydricity. The research aimed to investigate the effects of GO on the regeneration process and genetic stability of white strawberry plants and to use molecular markers to ensure that plants propagated in vitro are true to type. For this purpose, shoot tip explants were used and different concentrations of GO (0, 2.5, 5.0, 7.5, 10 mg/L) were added to the Murashige and Skoog (MS) medium for six weeks. The results showed that the optimum concentration for promoting the development of white strawberry seedlings was 7.5 mg/L of GO. The study also revealed that the addition of 7.5 mg/L GO in combination with 8 µM TDZ to the MS medium facilitated the induction of multiple shoots. Moreover, the clonal fidelity of the in vitro plants treated with GO showed a genetic similarity of over 97%. These results confirm that lower GO concentrations improve plant development and stability. Consequently, this nanomaterial has a positive effect on the growth of strawberry plants and is therefore well suited for strawberry tissue culture.

Deep Learning Assisted Surface-Enhanced Raman Spectroscopy (SERS) for Rapid and Direct Nucleic Acid Amplification and Detection: Toward Enhanced Molecular Diagnostics.

Surface-enhanced Raman scattering (SERS) has evolved into a robust analytical technique capable of detecting a variety of biomolecules despite challenges in securing a reliable Raman signal. Conventional SERS-based nucleic acid detection relies on hybridization assays, but reproducibility and signal strength issues have hindered research on directly amplifying nucleic acids on SERS surfaces. This study introduces a deep learning assisted ZnO-Au-SERS-based direct amplification (ZADA) system for rapid, sensitive molecular diagnostics. The system employs a SERS substrate fabricated by depositing gold on uniformly grown ZnO nanorods. These nanorods create hot spots for the amplification of the target nucleic acids directly on the SERS surface, eliminating the need for postamplification hybridization and Raman reporters. The limit of detection of the ZADA system was superior to those of the conventional amplification methods. Clinical validation of the ZADA system with coronavirus disease 2019 (COVID-19) samples from human patients yielded a sensitivity and specificity of 92.31% and 81.25%, respectively. The integration of a deep learning program further enhanced sensitivity and specificity to 100% and reduced SERS analysis time, showcasing the potential of the ZADA system for rapid, label-free disease diagnosis via direct nucleic acid amplification and detection within 20 min.

Evaluating direct amplification from viral transport medium for SARS-CoV-2 detection, strain typing, and angiotensin-converting enzyme genotyping and expression assays.

OBJECTIVE: The aim of this study was to compare the performance of direct amplification of viral nucleic acid from transport medium to extracted nucleic acid for polymerase chain reaction (PCR), sequencing, and genotyping applications. METHODS: XpressAmp lysate and extracted total nucleic acid from viral transport medium containing nasopharyngeal specimens were evaluated across different molecular applications to determine performance characteristics. RESULTS: SARS-CoV-2 quantitative PCR and angiotensin-converting enzyme (ACE) genotyping assays worked well with XpressAmp lysate, almost equal with or better than extracted nucleic acid in some specimens. However, XpressAmp completely failed to perform in next-generation sequencing for strain typing. Both protocols failed to detect ACE2 expression in viral transport medium. CONCLUSION: Direct amplification of viral nucleic acid from viral transport medium containing nasopharyngeal specimen works well for molecular assays with low thresholds of quality; however, it does have limitations with assays that require high quality nucleic acid for input. Use of the XpressAmp protocol significantly improves turnaround time and allows for easy ramp-up of PCR and genotyping assays.

Amplification of Extracted DNA and Direct Amplification with the PowerPlex® Y23 System.

The PowerPlex® Y23 System offered by Promega Corporation contains 23 Y-STR loci (DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and Y-GATA-H4). The PowerPlex® Y23 System is designed to amplify DNA from purified extracts as well as direct amplification from substrates used to collect database samples (e.g., swabs and storage cards). Protocols are provided for full-volume reactions for DNA extracts, as well as half-volume reactions for direct amplifications from different substrates.

QIAGEN's Investigator® 24plex QS and GO! PCR Amplification.

The Investigator® 24Plex kits are multiplex PCR kits utilized by forensic laboratories to simultaneously amplify 22 of the most commonly utilized STR markers for human identity testing, including the 20 core CODIS loci, along with the sex marker Amelogenin and 2 novel quality sensors. These quality sensors are unique internal PCR controls that provide useful insight to the analyst regarding possible inhibition or degradation within the sample. This chapter describes the use of the QS version of the kit designed for use with extracted DNA from casework samples, as well as the use of the GO! version of the kit designed for direct amplification of reference samples.

Alternative direct-to-amplification sperm cell lysis techniques for sexual assault sample processing.

The prevalence of sexual assault cases and increasingly sensitive DNA analysis methods have resulted in sexual assault kit backlogs in the United States. Although traditional DNA extraction and purification utilizing detergents, proteinase K, and DTT have been the primary technique for lysing sperm cell fractions from these samples, it is labor-intensive and inefficient regarding time and sperm DNA recovery - hindering the ability of forensic analysts to keep pace with evidence submissions. Thus, this study examined seven alternative sperm cell lysis techniques to develop a method that could efficiently lyse sperm and consistently generate high-quality profiles while also reducing time, labor, and cost requirements. Microscopic examination of lysates indicated only Casework Direct and alkaline techniques could lyse all spermatozoa within samples, while quantification results demonstrated all methods performed comparably to the control method of forensicGEM™ Sperm (p > 0.06). Amplification with 0.25 ng DNA revealed that unpurified lysates from Casework Direct, alkaline, and NP-40 techniques produced DNA profiles with acceptable mean STR peak heights and interlocus balance, both of which were similar to or better than the control. Overall, this study demonstrated the ability of Casework Direct, alkaline, and NP-40 methods to efficiently lyse spermatozoa and provide high-quality STR profiles despite the absence of a purification step. Ultimately, based on the data reported herein, alkaline lysis is the recommended alternative sperm lysis approach given its ability to generate high-quality profiles, save time, and decrease the cost per reaction when compared to traditional sperm cell lysis methods.

Inhibition of DNA amplification caused by metal in extracted bloodstains and in direct amplification.

Copper is a metal that is commonly found in many evidence types, such as weapons, and household items, that are often retrieved from crime scenes. We used copper and brass sheets, cartridge cases, and metal décor items as substrates for deposition of blood to study inhibition induced by metals. Another objective was to compare the efficiency of STR profiling following extraction, quantification, amplification, and capillary electrophoresis with a direct amplification method which only requires amplification and capillary electrophoresis. Samples that were designated for extraction, were collected, extracted, and purified using the Qiagen EZ1 DNA Investigator® Kit. Direct amplification was performed using Copan microFLOQ® Direct Swabs to collect minute amount of blood. Complete STR profiles were obtained from 54% of the extracted samples and from 88% of directly amplified samples. Substrate type and amplification method (two-way ANOVA, F(5, 131) = 3.59, p = 4.50 × 10-3) impacted profile completeness but collection time did not (one-way ANOVA, F(1, 131) = 0.017, p = 0.90). Composition of the substrates was determined with scanning electron microscopy (SEM), and inhibition caused by copper was evaluated for each substrate. When substrate composition was analyzed in combination with amplification method, a substrate's copper percent was not associated with increased allelic dropout (one-way ANOVA, F(1, 176) = 0.70, p = 0.40) but amplification alone was statistically significant (one-way ANOVA, F(1, 176) = 40.64, p = 1.56 × 10-9). Using the Copan microFLOQ® Direct Swabs, inhibition induced by metal observed was not as pronounced. In addition, less variability in profile completeness was detected, profiles were generated within a very short period, and only minute amount of bloodstain was needed, allowing for additional analysis of the same samples. Our results directly quantify the effect of copper composition on DNA profiling and suggest that while copper percentage is an observational contributor to allele dropout it is not wholly responsible for these events.

Collection and storage of DVI samples with microFLOQ® Direct swabs for direct amplification.

The rapid identification of decomposing human remains is a crucial component of disaster victim identification (DVI), often occurring in remote areas without access to laboratory or storage facilities. Due to the ease of collection and amenability to storage in harsh conditions, swabs may be used to collect DNA from decomposing remains as an alternative to sampling tissue or bone. Direct amplification could further streamline the process and reduce costs. This study investigated the efficacy of direct amplification of DVI samples using microFLOQ® Direct swabs and the QIAGEN Investigator QS GO! Kit. A comparison of performance between direct amplification and traditional methods was made to assess whether direct amplification offered an improvement to traditional methods. DNA was collected by swabbing the muscle of a decomposing human cadaver using three swab types (ADS Genetics 4N6FLOQSwabs®, NADS Genetics 4N6FLOQSwabs®, and the microFLOQ® Direct swab). Traditional swabs (4N6FLOQSwabs®) were extracted and quantified, while a direct amplification strategy was used with the microFLOQ® Direct swabs coupled with the Investigator 24Plex GO! Kit. Processing of the microFLOQ® Direct swabs were optimized and a hybrid strategy that used 4N6FLOQSwabs® to collect and store DNA before swabbing or "subsampling" the 4N6FLOQSwabs® for processing with microFLOQ® Direct swabs was developed. This hybrid strategy allowed for rapid processing without the consumption of the original sample. Traditional and direct PCR methods were comparable up to day 10 of decomposition depending on the sample location and for up to 3 months of storage at room temperature. This research indicated that microFLOQ® Direct swabs in conjunction with the Investigator 24Plex GO! Kit can be used to facilitate rapid direct processing of DNA from decomposing human remains.

Direct Amplification of Whole Blood and Amniotic Fluid Specimens for Prenatal and Postnatal Diagnosis of Hb E-ß 0-Thalassemia Diseases.

OBJECTIVE: Prenatal and postnatal diagnosis of hemoglobin E-ß 0-thalassemia can be made using polymerase chain reaction (PCR) analysis mostly on purified DNA. We have establihed a direct amplification method without DNA extraction on whole blood (WB) and amniotic fluid (AF) specimens to diagnose the disease. METHODS: Three reactions of WB PCR assays and 7 reactions of AF PCR tests were developed for postnatal and prenatal diagnosis, respectively. Assays were validated against routine tests in a blinded trial. RESULTS: The results showed 100% concordance with routine DNA PCR assays. Among 309 ß-thalassemia carriers, 191 patients (61.8%) carried common ß-thalassemia mutations. Among 448 AF specimens, 116 (25.9%) fetuses were found to be affected, 247 (55.1%) fetuses were carriers, and 85 (19%) fetuses were unaffected. CONCLUSION: We found that WB and AF PCR assays are simple, rapid, and reliable. The developed techniques could be applicable in routine settings.

The effects of dithiothreitol (DTT) on fluorescent qPCR dyes.

DNA extractions of semen samples commonly utilize dithiothreitol (DTT) to reduce and disrupt disulfide bonds. Although traditional extraction techniques remove DTT before downstream analyses, the forensic DNA community has recently explored Y-screening, direct amplification, and direct cell lysis assays that omit purification but employ reducing agents to lyse spermatozoa. This study examined the impact of residual DTT on downstream processes involving fluorescent dyes. Quantification using Investigator® Quantiplex HYres revealed a significant increase in the male DNA yield (p = 0.00056) and a >150,000,000-fold increase in the male:human DNA ratio when DTT remained in extracts versus when it was filtered out using a traditional purification method. When DTT was present with Quantifiler™ Trio, the true mean DNA yield for the large autosomal target significantly increased (p = 0.038) and the average reported DNA yields increased 1.1-fold, >9.5-fold, and 1.3-fold for the small autosomal, large autosomal, and male targets, respectively. DTT-spiked DNA standards from both kits were impacted similarly to samples with residual DTT, demonstrating that observed effects were related to DTT and not the extraction method. This study corroborates other reports that DTT adversely affects multiple dyes (e.g., Cy5, Quasar 670, SYBR Green I, TMR, and Mustang Purple® ). Overall, DTT causes inaccurate quantities and, consequently, inaccurate calculated male:female ratios when used in conjunction with these kits. Thus, implementation of newer direct-to-PCR assays incorporating DTT should either be avoided or used only with carefully evaluated, compatible dyes.

Collection and direct amplification methods using the GlobalFiler™ kit for DNA recovered from common pipe bomb substrates.

When analyzing DNA from exploded pipe bombs, quantities are often in trace amounts, making DNA typing extremely difficult. Amplifying minute amounts of DNA can cause stochastic effects resulting in partial or uninterpretable profiles. Therefore, the initial DNA collection from "touch" evidence must be optimized to maximize the amount of DNA available for analysis. This proof-of-concept study evaluated two different swab types with two direct amplification strategies to identify the most effective method for recovering DNA from common pipe bomb substrates. PVC and steel pipes, electrical tape, and copper wire spiked with epithelial cells were swabbed with cotton or microFLOQ® Direct Swabs and amplified directly or via a pre-treatment prior to STR amplification. Not only was the microFLOQ® Direct Swab protocol the quickest method with the least risk of contamination, but in combination with direct amplification, the microFLOQ® Direct Swabs also generated the most complete STR profiles.

Developmental validation of the Huaxia™ Platinum PCR amplification kit: A 6-dye multiplex direct amplification assay designed for Chinese reference samples.

The Huaxia™ Platinum Kit for short tandem repeat (STR) amplification was designed to meet the needs of the rapidly growing Chinese forensic database. This PCR multiplex allows simultaneous amplification of the following autosomal loci: D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, Penta E, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, D6S1043, D10S1248, D1S1656, D12S391, D2S1338, Penta D and the gender-identification markers Yindel, and AMEL. The Huaxia™ Platinum Kit enables direct amplification from blood and buccal samples stored on treated and untreated paper, and features an optimized PCR protocol that yields time to results in less than 45 min. Developmental validation testing followed SWGDAM guidelines and demonstrated that this assay produces reproducible and accurate results. Studies on 798 individuals in 4 major Chinese ethnic groups produced highly concordant results with other commercially available STR genotyping kits. The validation results demonstrate that the Huaxia™ Platinum Kit is a robust and reliable identification system for forensic DNA databasing applications.

Direct or DNA Extraction-Free Amplification and Quantification of Foodborne Pathogens.

The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.

A novel FTA™ elute card collection method that improves direct DNA amplification from bloodstained concrete.

Concrete is a common construction material found in residential and commercial buildings, bridges and parking lots that is a composite matrix containing aggregate held together with cement. The porous nature of concrete can make the collection and genotyping of biological fluids, such as blood, challenging. Forensic evidence can become embedded within the matrix, potentially reducing the amount of DNA available for analysis. In forensic science, "direct" amplification refers to a genotyping method that amplifies a DNA profile directly from a sample without DNA extraction, saving time and money. We investigated a novel application of Whatman™ FTA™ Elute cards in their ability to directly amplify PowerPlex® Fusion and Y23 profiles from minute amounts of blood that had been deposited on different concrete structures. In comparison to traditional collection methods, directly profiling blood stained construction materials using FTA™ Elute cards increased the percentage loci amplified and significantly improved both allele peak height and peak height ratio while reducing allelic drop-out. FTA™ Elute cards can provide a reliable, inexpensive and superior alternative to traditional methods.

[Individual Identification of Cartilage by Direct Amplification in Mass Disasters].

OBJECTIVES: To explore the effectiveness of direct amplification for the STR analysis of cartilage, and to accelerate the effectiveness of disaster victim identification. METHODS: Eighty-eight cartilage samples were directly amplified by PowerPle® 21 kit, and the results of genotyping were compared with that obtained by the magnetic beads method. RESULTS: In 88 cartilage samples, the STR genotypes were successfully detected from 84 samples by direct amplification and magnetic beads method, and both the results of genotyping by two method were consistent. CONCLUSIONS: Direct amplification with PowerPlex® 21 kit can be used for STR genotyping of cartilages. This method is operated easily and promptly, which has a potential application in the individual identification of mass disasters.

Quantification of microRNAs directly from body fluids using a base-stacking isothermal amplification method in a point-of-care device.

MicroRNAs have been proposed to be a class of biomarkers of disease as expression levels are significantly altered in various tissues and body fluids when compared to healthy controls. As such, the detection and quantification of microRNAs is imperative. While many methods have been established for quantification of microRNAs, they typically rely on time consuming handling such as RNA extraction, purification, or ligation. Here we describe a novel method for quantification of microRNAs using direct amplification in body fluids without upstream sample preparation. Tested with a point-of-care device (termed Gene-Z), the presence of microRNA promotes base-stacking hybridization, and subsequent amplification between two universal strands. The base-stacking approach, which was achieved in <60 min, provided a sensitivity of 1.4 fmol per reaction. Tested in various percentages of whole blood, plasma, and faeces, precision (coefficient of variation = 2.6%) was maintained and comparable to amplification in pristine samples. Overall, the developed method represents a significant step towards rapid, one-step detection of microRNAs.

Developmental validation of QIAGEN Investigator® 24plex QS Kit and Investigator® 24plex GO! Kit: Two 6-dye multiplex assays for the extended CODIS core loci.

The original CODIS database based on 13 core STR loci has been overwhelmingly successful for matching suspects with evidence. In order to increase the power of discrimination, reduce the possibility of adventitious matches, and expand global data sharing, the CODIS Core Loci Working Group determined the expansion of the CODIS core loci to 20 STR plus three additional "highly recommended" loci (SE33, DY391, Amelogenin) Hares, 2015, 2012 [1,2]. The QIAGEN Investigator 24plex QS and Investigator 24plex GO! Kits are 6-dye multiplex assays that contain all markers of the expanded 23 CODIS core loci along with a unique internal performance control that is co-amplified with the STR markers. The "Quality Sensor" generates additional information for quality control and performance checks. Investigator 24plex QS is designed for purified DNA from casework and reference samples, whereas 24plex GO! is dedicated to direct amplification of reference samples, like blood or buccal cells on FTA or swabs. A developmental validation study was performed on both assays. Here, we report the results of this study which followed the recommendations of the European Network of Forensic Science Institutes (ENFSI) [3] and the Revised Validation Guidelines of the Scientific Working Group on DNA Analysis Methods (SWGDAM) [4]. Data included are for PCR-based procedures e.g. reaction conditions, effects of PCR annealing temperature variations, amplification cycles or cyclers, sensitivity (also in the context of the Quality Sensor), performance with simulated inhibition, stability and efficiency, precision, reproducibility, mixture study, concordance, stutter, species specificity, and case-type samples. The validation results demonstrate that the Investigator 24plex QS and Investigator 24plex GO! Kits are robust and reliable identification assays as required for forensic DNA typing in forensic casework analysis and databasing.

Evaluation of Nucleic Acid Isothermal Amplification Methods for Human Clinical Microbial Infection Detection.

Battling infection is a major healthcare objective. Untreated infections can rapidly evolve toward the condition of sepsis in which the body begins to fail and resuscitation becomes critical and tenuous. Identification of infection followed by rapid antimicrobial treatment are primary goals of medical care, but precise identification of offending organisms by current methods is slow and broad spectrum empirical therapy is employed to cover most potential pathogens. Current methods for identification of bacterial pathogens in a clinical setting typically require days of time, or a 4- to 8-h growth phase followed by DNA extraction, purification and PCR-based amplification. We demonstrate rapid (70-120 min) genetic diagnostics methods utilizing loop-mediated isothermal amplification (LAMP) to test for 15 common infection pathogen targets, called the Infection Diagnosis Panel (In-Dx). The method utilizes filtration to rapidly concentrate bacteria in sample matrices with lower bacterial loads and direct LAMP amplification without DNA purification from clinical blood, urine, wound, sputum and stool samples. The In-Dx panel was tested using two methods of detection: (1) real-time thermocycler fluorescent detection of LAMP amplification and (2) visual discrimination of color change in the presence of Eriochrome Black T (EBT) dye following amplification. In total, 239 duplicate samples were collected (31 blood, 122 urine, 73 mucocutaneous wound/swab, 11 sputum and two stool) from 229 prospectively enrolled hospital patients with suspected clinical infection and analyzed both at the hospital and by In-Dx. Sensitivity (Se) of the In-Dx panel targets pathogens from urine samples by In-Dx was 91.1% and specificity (Sp) was 97.3%, with a positive predictive value (PPV) of 53.7% and a negative predictive value (NPV) of 99.7% as compared to clinical microbial detection methods. Sensitivity of detection of the In-Dx panel from mucocutaneous swab samples was 65.5% with a Sp of 99.3%, and a PPV of 84% and NPV of 98% as compared to clinical microbial detection methods. Results indicate the LAMP-based In-Dx panel allows rapid and precise diagnosis of clinical infections by targeted pathogens across multiple culture types for point-of-care utilization.