Evaluation of reverse transcription yield of RNA standards and forensic samples based on droplet digital PCR.

RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 µg/µL - 0.24 ng/µL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 µL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.

Assessment of the efficiency of DNA isolation and profiling applying a temperature-driven method in human remains.

The identification of human remains is of utmost importance in a variety of scenarios. One of the primary identification methods is DNA. DNA extraction from human remains could be difficult, particularly in situations where the remains have been exposed to environmental conditions and other insults. Several studies tried to improve extraction by applying different approaches. ForensicGEM Universal (MicroGem) is a single-tube approach to DNA extraction and a temperature-driven method that could have some advantages with respect to previous techniques, among them, reducing the risk of contamination, not requiring specialized equipment, or several steps to perform. The aim of this study was to assess, for the first time, the efficiency of DNA extraction and quality of STR profiles applying the MicroGem protocol and modifications of this protocol from tooth samples in comparison with automatic extraction (AE). Our results indicated that AE and MicroGem performed similar, though with variability depending on the MicroGem modifications, increasing the DNA yield and STR profile quality when DNA is concentrated with Microcon. These findings demonstrated the efficiency of this methodology for DNA extraction from human remains while also providing a simple and quick technique suitable to apply in a variety of forensic scenarios.

A rapid and low-cost platform for detection of bacterial based on microchamber PCR microfluidic chip.

Polymerase chain reaction (PCR) has been considered as the gold standard for detecting nucleic acids. The simple PCR system is of great significance for medical applications in remote areas, especially for the developing countries. Herein, we proposed a low-cost self-assembled platform for microchamber PCR. The working principle is rotating the chamber PCR microfluidic chip between two heaters with fixed temperature to solve the problem of low temperature variation rate. The system consists of two temperature controllers, a screw slide rail, a chamber array microfluidic chip and a self-built software. Such a system can be constructed at a cost of about US$60. The micro chamber PCR can be finished by rotating the microfluidic chip between two heaters with fixed temperature. Results demonstrated that the sensitivity of the temperature controller is 0.1℃. The relative error of the duration for the microfluidic chip was 0.02 s. Finally, we successfully finished amplification of the target gene of Porphyromonas gingivalis in the chamber PCR microfluidic chip within 35 min and on-site detection of its PCR products by fluorescence. The chip consisted of 3200 cylindrical chambers. The volume of reagent in each volume is as low as 0.628 nL. This work provides an effective method to reduce the amplification time required for micro chamber PCR.

Bridging Flocculation of a Sterically Stabilized Cationic Latex as a Biosensor for the Detection of Microbial DNA after Amplification via PCR.

There is a high demand for rapid, sensitive, and accurate detection methods for pathogens. This paper demonstrates a method of detecting the presence of amplified DNA from a range of pathogens associated with serious infections including Gram-negative bacteria, Gram-positive bacteria, and viruses. DNA is amplified using a polymerase chain reaction (PCR) and consequently detected using a sterically stabilized, cationic polymer latex. The DNA induces flocculation of this cationic latex, which consequently leads to rapid sedimentation and a visible change from a milky-white dispersion to one with a transparent supernatant, presenting a clear visible change, indicating the presence of amplified DNA. Specifically, a number of different pathogens were amplified using conventional or qPCR, including Staphylococcus aureus, Escherichia coli, and Herpes Simplex Virus (HSV-2). This method was demonstrated to detect the presence of bacteria in suspension concentrations greater than 380 CFU mL-1 and diagnose the presence of specific genomes through primer selection, as exemplified using methicillin resistant and methicillin susceptible Staphylococcus aureus. The versatility of this methodology was further demonstrated by showing that false positive results do not occur when a PCR of fungal DNA from C. albicans is conducted using bacterial universal primers.

Rapid and inexpensive method of PCR ready DNA isolation from human peripheral blood and saliva.

BACKGROUND: The isolation of nucleic acids is a frequently performed procedure in the molecular biology area. Although several rapid DNA isolation techniques from human peripheral blood and saliva have been developed, there are still some disadvantages - volume, time, cost, and yield are a few notable ones. OBJECTIVE: We aim to develop a rapid and inexpensive method to isolate high-molecular-weight genomic DNA from human peripheral blood and saliva that can be used for molecular biology experiments. METHODS: Five DNA isolation methods with slightly varying protocols were used. High-quality DNA obtained from one specific method was further amplified by PCR and the template with good amplification was further used for performing RFLP and sequencing. RESULTS: Out of 5 different isolation methods (R1 to R5), DNA obtained from the R4 was of good quality (molecular weight is > 10 kb and 260/280 ratio is 1.89 ± 0.2), which allows successful PCR amplification and good separation in Restriction Fragment Length Polymorphism analysis. Sequencing by the Sanger Sequencing produced a good readable sequence of an amplified fragment from Method R4 DNA. CONCLUSION: In the present study we have developed a simple, rapid, and cost-effective DNA isolation method, which uses low sample volume and yields good quantity and high-quality product. The DNA obtained is highly fit for molecular genetics research applications.

Polydopamine nanospheres-assisted direct PCR for rapid detection of Escherichia coli O157:H7.

Polymerase chain reaction (PCR) is one of the most common methods for rapid monitoring of foodborne pathogens; however, it requires purified nucleic acid as a template. Conventional nucleic acid purification is a time-consuming and laborious process. To overcome this, we developed polydopamine nanospheres (PDA NPs)-assisted direct PCR for detecting Escherichia coli O157:H7 (E. coli O157:H7). PDA NPs significantly enhanced PCR efficiency because of their strong interaction with PCR reagents, including polymerase and primers, thereby enabling regulation of the PCR performance. The optimal concentration and diameter for PDA NPs were 0.10 µg/µL and 504 nm, respectively. The PDA NPs-assisted direct PCR exhibited high sensitivity in E.coli O157:H7 detection. The detection limit of PDA NPs-assisted direct PCR was 6.7 × 104 CFU/mL, which was 10-fold lower than that of direct PCR (6.7 × 105 CFU/mL). Moreover, the sensor demonstrated excellent selectivity against E. coli O157:H7, with a negative reaction to eight other common pathogens. Most importantly, the PDA NPs-assisted direct PCR detected the order of 104-5 CFU/mL E.coli O157:H7 in milk, beef, and watermelon samples. No cultural enrichment was required, with the whole process taking <3 h. Therefore, PDA NPs-assisted direct PCR has tremendous potential in the rapid and sensitive detection of pathogens.

An effective method for saliva stabilization and magnetic nanoparticles based DNA extraction for genomic applications.

Introduction of magnetisable solid phase extraction procedures have provided various advantages over spin-column based extraction techniques. Although certain methods for magnetic bead based extraction of DNA from human saliva already exist, there is still a need to address the inadequate purity profile and low yield which occur due to the inefficiency of extraction methods. Hence, an improved method for DNA extraction from human saliva using uncoated magnetic nanoparticles (MNPs) intended to resolve the issues mentioned above is described here. The uncoated magnetic nanoparticles used in this study facilitate reversible binding of DNA and due to the absence of surface coating the particle size remains small thereby providing higher surface area to volume ratio for binding DNA. Another objective of this study was to develop a saliva preservation buffer (SPB) to solve the major challenges associated with storage and easy transportation of saliva sample at room temperature. Human saliva samples stored in the saliva preservation buffer were stable up to 160 days at room temperature without any bacterial or fungal growth and the quality of genomic DNA was intact.

Construction and characterization of an infectious cDNA clone of enterovirus 71: a rapid method for rescuing infectious virus based on stable cells expressing T7 polymerase.

Enterovirus 71 (EV71) is a causative agent of hand, foot and, mouth disease (HFMD) in young children. It is valuable for virologists to develop a fast method to rescue infectious virus from a viral cDNA clone. Here, we report a method for rapid rescue of infectious EV71 by using cells expressing T7 polymerase. The full-length EV71 genome was amplified in one step by long-distance PCR with a T7 promoter at the 5' end, and the T7 polymerase gene was cloned into a lentivirus vector for construction of a stable cell line expressing T7 polymerase. The infectious virus was rapidly and efficiently rescued by single transfection of cells with the infectious cDNA clone. Further experiments showed that the rescued virus had characteristics similar to those of the parental virus. This method circumvented the difficulty in performing in vitro transcription of a long linear DNA to obtain high-quality RNA. The construction of the viral cDNA clone and the fast rescue of the infectious virus will greatly benefit future investigations.

The relationship between the simultaneity present of cagA and hopQI genes in Helicobacter pylori and the risk of gastric cancer.

Helicobacter pylori is a bacterium that causes infections in the gastrointestinal tract. This type of bacterium is very common and contagious at the same time. H. pylori enters the mouth and continues its course along the gastrointestinal tract. H. pylori infection induces an inflammatory response that leads to the activity of neutrophils, lymphocytes, plasma cells, and macrophages. In addition to the bacterial role in gastric mucosa, the host's inflammatory response may also play a role in disease outcome. In inflammation, the risk of carcinogenesis increases due to DNA damage increased proliferation and the creation of an environment rich in cytokines and growth factors. Genetic methods and diagnosis of H. pylori genes are used to identify healthy and healthy gastric cancer patients infected with H. pylori. In relation to the genes associated with H. pylori pathogenesis, the presence of genes such as cagA, hopQI, hopQII and so on is used, and PCR of a part of these genes amplified fragments of different lengths. One of the less-studied cases is the association of two or more pathogenic genes simultaneously with H. pylori. In this research, the frequency of disease and healthy individuals who are infected with H. pylori and have two genotypes cagA and hopQI at the same time, was examined. In order to diagnose H. pylori-infected individuals in healthy and gastric cancer patients, after PCR of glmM gene, PCR product electrophoresis on agarose gel was used. For this purpose, gastric tissue biopsy was used in patients and saliva was used in healthy individuals. For this purpose, 100 gastric biopsy samples were collected from patients with gastric cancer and 100 saliva samples from healthy individuals. According to the data, there is a significant relationship between the simultaneous presence of two genes cagA and hopQI and gastric cancer. In patients, 45.3% showed both genotypes, while in healthy individuals only 10.5% have this genotype and other healthy but infected with H. pylori (90.8%) do not have this genotype. To be. No report was observed on the simultaneous study of cagA and hopQI genes. No report was observed regarding the simultaneous study of cagA and hopQI genes.

Rapid in vitro production of single-stranded DNA.

There is increasing demand for single-stranded DNA (ssDNA) of lengths >200 nucleotides (nt) in synthetic biology, biological imaging and bionanotechnology. Existing methods to produce high-purity long ssDNA face limitations in scalability, complexity of protocol steps and/or yield. We present a rapid, high-yielding and user-friendly method for in vitro production of high-purity ssDNA with lengths up to at least seven kilobases. Polymerase chain reaction (PCR) with a forward primer bearing a methanol-responsive polymer generates a tagged amplicon that enables selective precipitation of the modified strand under denaturing conditions. We demonstrate that ssDNA is recoverable in ∼40-50 min (time after PCR) with >70% yield with respect to the input PCR amplicon, or up to 70 pmol per 100 µl PCR reaction. We demonstrate that the recovered ssDNA can be used for CRISPR/Cas9 homology directed repair in human cells, DNA-origami folding and fluorescent in-situ hybridization.

PCR array analysis identified hyperproliferation but not autophagy or apoptosis in fibrous epulis.

BACKGROUND: The pathogenesis of fibrous epulis is still quite unclear. Our recent genome-wide RNA sequencing analysis revealed that in fibrous epulis, RAS-PI3K-AKT-NF-κB pathway regulates the expression of Bcl-2 family and IAP family genes, leading to increased proliferation and the inhibition of apoptosis. The PI3K/AKT signaling pathway can promote autophagy in human gingival fibroblasts; therefore, the purpose of the present study was to identify whether autophagy is involved in the pathogenesis of fibrous epulis. METHODS: Differentially expressed genes (DEGs) between fibrous epulis lesions and normal gingival tissues were identified using the PCR array. The expression levels of eighteen autophagy-related (ATG) family genes, twelve B-cell lymphoma 2 (Bcl-2) family genes, and eleven cysteine-dependent aspartate-directed protease (caspase) family genes were validated using quantitative real-time PCR (qRT-PCR). Autophagy induction was determined by measuring microtubule-associated protein light chain 3 (LC3) conversion (LC3-I to LC3-II) by immunoblot analysis. RESULTS: The PCR array identified six upregulated genes, whereas no genes were expressed at significantly lower levels. The upregulated genes were BCL2, BCL2L1, CXCR4, HSP90AA1, HSPA8, and IGF1, which all belong to the "regulation of autophagy" group but not the "autophagy machinery components" group. qRT-PCR verified that the expression levels of BCL2, BCL2L1 (also known as BCL-XL), and BCL2L2 (also known as BCL-W) were significantly increased in fibrous epulis. No LC3-I to LC3-II conversion was observed. CONCLUSIONS: The present study reveals that in fibrous epulis, Bcl-2 and Bcl-xL coordinately mediate gingival cell escape from apoptosis, leading to uncontrolled proliferation. Moreover, ATG family genes are not activated, and autophagy is not involved in this process.

Antibody detection by agglutination-PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis.

Oral fluid (OF) is a highly effective substrate for population-based HIV screening efforts, as it is noninfectious and significantly easier to collect than blood. However, anti-HIV antibodies are found at far lower concentrations in OF compared with blood, leading to poor sensitivity and a longer period of time from infection to detection threshold. Thus, despite its inherent advantages in sample collection, OF is not widely used for population screening. Here we report the development of an HIV OF assay based on Antibody Detection by Agglutination-PCR (ADAP) technology. This assay is 1,000-10,000 times more analytically sensitive than clinical enzyme-linked immunoassays (EIAs), displaying both 100% clinical sensitivity and 100% specificity for detecting HIV antibodies within OF samples. We show that the enhanced analytical sensitivity enables this assay to correctly identify HIV-infected individuals otherwise missed by current OF assays. We envision that the attributes of this improved HIV OF assay can increase testing rates of at-risk individuals while enabling diagnosis and treatment at an earlier time point.

Characterization of the Streptococcus mutans SMU.1703c-SMU.1702c Operon Reveals Its Role in Riboflavin Import and Response to Acid Stress.

Streptococcus mutans utilizes numerous metabolite transporters to obtain essential nutrients in the "feast or famine" environment of the human mouth. S. mutans and most other streptococci are considered auxotrophic for several essential vitamins including riboflavin (vitamin B2), which is used to generate key cofactors and to perform numerous cellular redox reactions. Despite the well-known contributions of this vitamin to central metabolism, little is known about how S. mutans obtains and metabolizes B2 The uncharacterized protein SMU.1703c displays high sequence homology to the riboflavin transporter RibU. Deletion of SMU.1703c hindered S. mutans growth in complex and defined medium in the absence of saturating levels of exogenous riboflavin, whereas deletion of cotranscribed SMU.1702c alone had no apparent effect on growth. Expression of SMU.1703c in a Bacillus subtilis riboflavin auxotroph functionally complemented growth in nonsaturating riboflavin conditions. S. mutans was also able to grow on flavin adenine dinucleotide (FAD) or flavin mononucleotide (FMN) in an SMU.1703c-dependent manner. Deletion of SMU.1703c and/or SMU.1702c impacted S. mutans acid stress tolerance, as all mutants showed improved growth at pH 5.5 compared to that of the wild type when medium was supplemented with saturating riboflavin. Cooccurrence of SMU.1703c and SMU.1702c, a hypothetical PAP2 family acid phosphatase gene, appears unique to the streptococci and may suggest a connection of SMU.1702c to the acquisition or metabolism of flavins within this genus. Identification of SMU.1703c as a RibU-like riboflavin transporter furthers our understanding of how S. mutans acquires essential micronutrients within the oral cavity and how this pathogen successfully competes within nutrient-starved oral biofilms.IMPORTANCE Dental caries form when acid produced by oral bacteria erodes tooth enamel. This process is driven by the fermentative metabolism of cariogenic bacteria, most notably Streptococcus mutans Nutrient acquisition is key in the competitive oral cavity, and many organisms have evolved various strategies to procure carbon sources or necessary biomolecules. B vitamins, such as riboflavin, which many oral streptococci must scavenge from the oral environment, are necessary for survival within the competitive oral cavity. However, the primary mechanism and proteins involved in this process remain uncharacterized. This study is important because it identifies a key step in S. mutans riboflavin acquisition and cofactor generation, which may enable the development of novel anticaries treatment strategies via selective targeting of metabolite transporters.

Diagnostic reliability of nested PCR depends on the primer design and threshold abundance of Helicobacter pylori in biopsy, stool, and saliva samples.

BACKGROUND: The aim of this work was to find a reliable nested PCR for the detection of Helicobacter pylori in biopsy, stool, and saliva specimens. MATERIALS AND METHODS: Novel nested PCR was elaborated and validated on 81 clinical biopsy, stool, and saliva samples from the same individual and compared to available H pylori assays: histology, rapid urease test (RUT), stool antigen test (SAT), 13 C-urea breath test (UBT). RESULTS: The efficiency and selectivity of 17 published nested polymerase chain reactions (PCR) available for Helicobacter pylori detection were re-evaluated. Most of them had serious limitations and mistakes in primer design. Hence, we elaborated a nested PCR for the unambiguous identification of H pylori in biopsy, stool, and saliva, using primers targeted to variable regions of the 16S ribosomal RNA (rRNA) gene. Moreover, we determined the detection limit by adding a known number of cells. This number was as low as 0.5 cells in a PCR vial, but due to the DNA isolation procedures, it required 1-5 × 103 cells/g or ml of specimen. The sensitivity for nested PCR from stomach biopsies was on the same scale as 13 C-UBT (93.8%), but it was much lower in amplifications from stool (31.3%). Sequencing of all obtained PCR products exclusively confirmed H pylori-specific DNA sequences. CONCLUSIONS: Elaborated nested PCR assay can serve as an auxiliary method for controversial samples (patients with bleeding or taking proton-pump inhibitor) in laboratories with basic equipment. The sensitivity and specificity for the amplification from gastric biopsies was almost like 13 C-UBT. Despite the good sensitivity, the threshold occurrence and the ability to survive in the oral cavity aside from and independent of the stomach is the reason why H pylori DNA cannot be reliably detected in saliva, stool, and some biopsy samples.

Validation of the AGCU Expressmarker 16 + 22Y Kit: a new multiplex for forensic application.

Genotyping by targeting short tandem repeats (STRs) has been widely used in forensic applications. However, most commercial kits detect autosomal STRs or Y-STRs alone, which waste both time and opportunity. The AGCU Expressmarker 16 + 22Y Kit includes 16 autosomal and 22 Y-chromosomal STR loci and is designed for the forensic science field and obtaining quicker results. Here, we conducted the validation study according to Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. Validation of PCR-based studies, species specificity, sensitivity, DNA mixture studies, inhibitors, precision, and sizing accuracy were performed. Furthermore, this system was also tested in 346 random male samples from Han, Hui, Tibetan, and Zhuang populations in China, showing its high power for forensic discrimination in the Chinese population. In addition, this system was able to deal with AMELY deletion cases, which can correctly identify sex in forensic criminal investigations. Our results suggested that the AGCU Expressmarker 16 + 22Y Kit is a useful tool for rapid criminal investigation.

An evaluation of direct PCR assays for the detection and quantification of Porphyromonas gingivalis.

Porphyromonas gingivalis has been linked to the development and progression of oesophageal squamous cell carcinoma (ESCC), and is considered to be a high-risk factor for ESCC. Currently, the commonly used methods for P. gingivalis detection are culture or DNA extraction-based, which are either time and labour intensive especially for high-throughput applications. We aimed to establish and evaluate a rapid and sensitive direct quantitative polymerase chain reaction (qPCR) protocol for the detection of P. gingivalis without DNA extraction which is suitable for large-scale epidemiological studies. Paired gingival swab samples from 192 subjects undergoing general medical examinations were analysed using two direct and one extraction-based qPCR assays for P. gingivalis. Tris-EDTA buffer-based direct qPCR (TE-direct qPCR), lysis-based direct qPCR (lysis-direct qPCR) and DNA extraction-based qPCR (kit-qPCR) were used, respectively, in 192, 132 and 60 of these samples for quantification of P. gingivalis. The sensitivity and specificity of TE-direct qPCR was 95.24% and 100% compared with lysis-direct qPCR, which was 100% and 97.30% when compared with kit-qPCR; TE-direct qPCR had an almost perfect agreement with lysis-direct qPCR (κ = 0.954) and kit-qPCR (κ = 0.965). Moreover, the assay time used for TE-direct qPCR was 1.5 h. In conclusion, the TE-direct qPCR assay is a simple and efficient method for the quantification of oral P. gingivalis and showed high sensitivity and specificity compared with routine qPCR.

Rapid and visual detection of porcine deltacoronavirus by recombinase polymerase amplification combined with a lateral flow dipstick.

BACKGROUND: Porcine Deltacoronavirus (PDCoV) is a newly emerging Coronavirus that was first identified in 2012 in Hong Kong, China. Since then, PDCoV has subsequently been reported worldwide, causing a high number of neonatal piglet deaths and significant economic losses to the swine industry. Therefore, it is necessary to establish a highly sensitive and specific method for the rapid diagnosis of PDCoV. RESULTS: In the present study, a highly sensitive and specific diagnostic method using recombinase polymerase amplification combined with a lateral flow dipstick (LFD-RPA) was developed for rapid and visual detection of PDCoV. The system can be performed under a broad range of temperature conditions from 10 to 37 °C, and the detection of PDCoV can be completed in 10 min at 37 °C. The sensitivity of this assay was 10 times higher than that of conventional PCR with a lower detection limit of 1 × 102 copies/µl of PDCoV. Meanwhile, the LFD-RPA assay specifically amplified PDCoV, while there was no cross-amplification with other swine-associated viruses, including Porcine epidemic diarrhea virus (PEDV), Transmissible gastroenteritis virus (TGEV), Porcine kobuvirus (PKoV), Foot and mouth disease virus (FMDV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV2), Classical swine fever virus (CSFV) and Seneca valley virus (SVV). The repeatability of the test results indicated that this assay had good repeatability. In addition, 68 clinical samples (48 fecal swab specimens and 20 intestinal specimens) were further tested by LFD-RPA and RT-PCR assay. The positive rate of LFD-RPA clinical samples was 26.47% higher than that of conventional PCR (23.53%). CONCLUSIONS: The LFD-RPA assay successfully detected PDCoV in less than 20 min in this study, providing a potentially valuable tool to improve molecular detection for PDCoV and to monitor the outbreak of PDCoV, especially in low-resource areas and laboratories.

Direct genotyping from whole blood using alkaline polyethylene glycol.

In order to simplify biological sample preparation to meet the demand of rapid genotyping, we improve alkaline polyethylene glycol (APEG) based on Chomczynski's procedure for the PCR-based lateral flow genotyping system, which enables the rapid and efficient direct genotyping from whole blood without DNA purification. The improved APEG has a high tolerance for extreme storage conditions. Testing whole blood with an abnormal hematological index indicates that APEG efficiency would not be influenced by pathological factors. Compared with sequencing, the accuracy of this genotyping system was 100% on testing with 200 clinical samples.

The effects of extra PCR cycles when amplifying skeletal samples with the GlobalFiler® PCR Amplification Kit.

When samples with low amounts of DNA are amplified using short tandem repeats (STRs), stochastic effects such as allele and locus dropout or drop-in, allele imbalance, and increased stutter often occur making data interpretation more difficult. The most common approach to improving STR results from low template samples is to increase the number of PCR cycles. Although more alleles may be recovered, stochastic effects may be exaggerated resulting in more complicated STR profiles. This work reports the effect of additional PCR cycles (29 vs. 30, 31, and 32) on STR success from environmentally challenged bone and tooth samples using the GlobalFiler® DNA Amplification Kit (Thermo Fisher Scientific). In addition, we compared the efficiency of two DNA extraction kits for skeletal samples: QIAamp® DNA Investigator (QIAGEN) and PrepFiler® BTA™ Forensic DNA Extraction (Thermo Fisher Scientific) kits. Results showed that more DNA was recovered from samples using the PrepFiler® BTA™ kit; but regardless of the extraction method, the number of alleles detected and the peak heights both increased with an increase in PCR cycle number. Although more alleles were reported in almost all samples, the most notable improvement was observed in samples with the DNA template < 120 pg. A general increase in the number of PCR artifacts was detected in STR profiles generated using 30-32 cycles. Overall, this study provides supporting evidence that STR profile completeness and quality may be improved when low template skeletal samples are amplified with extra PCR cycles (up to 32 cycles) using the GlobalFiler® DNA Amplification Kit.

A fast nucleic acid extraction system for point-of-care and integration of digital PCR.

Digital PCR is a powerful amplification method for absolute quantification of nucleic acids. The systems that integrated the nucleic acid extraction and amplification can reduce detection time, improve accuracy, and reduce labor costs. However, current nucleic acid extraction systems cannot be integrated well with integrated fluidic circuit (IFC) dPCR or droplet digital PCR chips perfectly and limit the application of digital PCR. In this study, a polytetrafluoroethylene (PTFE)-based nucleic acid extraction (PNE) system, which was able to achieve fully closed extraction for micro samples and was able to be integrated with IFC dPCR or droplet digital dPCR (ddPCR) chips perfectly is proposed. For this system, PTFE tubing with an inner diameter of 1 mm was used to load the reagents and superparamagnetic particles (PMPs) were used to extract nucleic acids. The system can extract nucleic acids from cells and blood in 5 minutes. Meanwhile, when nucleic acid extraction was completed, PNE was able to be directly combined with IFC dPCR or ddPCR chips without any intermediate steps. Therefore, the PNE system can realize sample-in-digital-answer-out. It will be highly useful in point-of-care (POC) and promote the development and application of dPCR.