Direct Amplification Research Articles

Modulating gene regulation function by chemically controlled transcription factor clustering

Recent studies have suggested that transcriptional protein condensates (or clusters) may play key roles in gene regulation and cell fate determination. However, it remains largely unclear how the gene regulation function is quantitatively tuned by transcription factor (TF) clustering and whether TF clustering may confer emergent behaviors as in cell fate control systems. Here, to address this, we construct synthetic TFs whose clustering behavior can be chemically controlled. Through single-parameter tuning of the system (i.e., TF clustering propensity), we provide lines of evidence supporting the direct transcriptional activation and amplification of target genes by TF clustering. Single-gene imaging suggests that such amplification results from the modulation of transcriptional dynamics. Importantly, TF clustering propensity modulates the gene regulation function by significantly tuning the effective TF binding affinity and to a lesser extent the ultrasensitivity, contributing to bimodality and sustained response behavior that are reminiscent of canonical cell fate control systems. Collectively, these results demonstrate that TF clustering can modulate the gene regulation function to enable emergent behaviors, and highlight the potential applications of chemically controlled protein clustering.

Selective Whole-Genome Amplification as a Tool to Enrich Specimens with Low Treponema pallidum Genomic DNA Copies for Whole-Genome Sequencing.

ABSTRACTDownstream next-generation sequencing (NGS) of the syphilis spirochete Treponema pallidum subspecies pallidum (T. pallidum) is hindered by low bacterial loads and the overwhelming presence of background metagenomic DNA in clinical specimens. In this study, we investigated selective whole-genome amplification (SWGA) utilizing multiple displacement amplification (MDA) in conjunction with custom oligonucleotides with an increased specificity for the T. pallidum genome and the capture and removal of 5′-C-phosphate-G-3′ (CpG) methylated host DNA using the NEBNext Microbiome DNA enrichment kit followed by MDA with the REPLI-g single cell kit as enrichment methods to improve the yields of T. pallidum DNA in isolates and lesion specimens from syphilis patients. Sequencing was performed using the Illumina MiSeq v2 500 cycle or NovaSeq 6000 SP platform. These two enrichment methods led to 93 to 98% genome coverage at 5 reads/site in 5 clinical specimens from the United States and rabbit-propagated isolates, containing >14 T. pallidum genomic copies/μL of sample for SWGA and >129 genomic copies/μL for CpG methylation capture with MDA. Variant analysis using sequencing data derived from SWGA-enriched specimens showed that all 5 clinical strains had the A2058G mutation associated with azithromycin resistance. SWGA is a robust method that allows direct whole-genome sequencing (WGS) of specimens containing very low numbers of T. pallidum, which has been challenging until now.IMPORTANCE Syphilis is a sexually transmitted, disseminated acute and chronic infection caused by the bacterial pathogen Treponema pallidum subspecies pallidum. Primary syphilis typically presents as single or multiple mucocutaneous lesions and, if left untreated, can progress through multiple stages with various clinical manifestations. Molecular studies often rely on direct amplification of DNA sequences from clinical specimens; however, this can be impacted by inadequate samples due to disease progression or timing of patients seeking clinical care. While genotyping has provided important data on circulating strains over the past 2 decades, WGS data are needed to better understand strain diversity, perform evolutionary tracing, and monitor antimicrobial resistance markers. The significance of our research is the development of an SWGA DNA enrichment method that expands the range of clinical specimens that can be directly sequenced to include samples with low numbers of T. pallidum.

Triplet-Primed PCR Assays for Accurate Screening of FMR1 CGG Repeat Expansion and Genotype Verification.

Fragile X syndrome and other fragile X-associated disorders are caused by the full-mutation (>200 copies) and premutation (55 to 200 copies) expansion, respectively, of the CGG short tandem repeat in the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Clinical diagnostic laboratories use Southern blot analysis and polymerase chain reaction (PCR)-based tests to detect and/or size the FMR1 CGG repeats. The development of sensitive and high-throughput triplet-primed PCR (TP-PCR) assays has diminished the need to subject all samples to Southern blot analysis, which is both labor- and time-intensive. In this article, we describe two direct TP-PCR (dTP-PCR) assays for the detection of FMR1 CGG repeat expansions. We outline a protocol that is based on melting curve analysis of dTP-PCR amplicons for a rapid and cost-effective first-tier screening and identification of individuals with premutation and full-mutation expansions. We also describe a protocol that employs capillary electrophoresis to resolve the dTP-PCR amplicon fragments and to estimate the repeat sizes of normal (5 to 44 copies), intermediate (45 to 54 copies), and premutation alleles, as well as to detect full mutations and determine the structure of the FMR1 alleles. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for melting curve analysis Basic Protocol 2: Melting curve analysis of direct triplet-primed PCR amplicons on the Rotor-Gene Q MD × 5plex high-resolution melt platform Alternate Protocol: Melting curve analysis of direct triplet-primed PCR amplicons on the LightCycler 480 system Basic Protocol 3: Generation of direct triplet-primed PCR melting curve analysis profiles Basic Protocol 4: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for capillary electrophoresis Basic Protocol 5: Generation of control FMR1 plasmids for direct triplet-primed PCR melting curve analysis Basic Protocol 6: Sanger sequencing assay to verify FMR1 CGG repeat size and structure of plasmid DNA controls.

On Optically Modulated Reflective Semiconductor Optical Amplifier Pattern-Dependent Overshoot Mitigation Using a Birefringent Fiber Loop

Reflective semiconductor optical amplifiers (RSOAs) are key elements for modern optical communications. Despite their widespread deployment, their performance when intended for ultrafast data amplification is limited by their inherently slow gain dynamics. In this paper, we propose to employ a birefringent fiber loop (BFL) to compensate for the RSOA pattern-dependent behavior and extend its operation well beyond that allowed by its nominal optical modulation bandwidth. We apply a reduced model to describe the RSOA response and quantify the RSOA output distortion by means of a non-return-to-zero data pulse overshoot. We validate the outcomes of this model in the time domain both for the RSOA alone and with the assistance of the BFL by an extensive comparison to available measurements. The excellent matching between simulation and experimental results allows us to further investigate the impact of critical operating parameters and derive specifications for them so that the performance of the scheme against the overshoot is made acceptable. The theoretical predictions confirm the ability of the BFL to enhance the RSOA direct amplification capability and hence establish it as a frequency discriminator for complementing RSOAs’ versatile and scalable operation.

Regulation of chloroplast ultrastructure, adventitious bud anatomy and clonal fidelity of in vitro Zingiber officinale var. rubrum Theilade plantlets in response to LED spectral quality

Light quality plays a major role in deciding plant physiological processes during growth and development as it interacts with the photoreceptors to induce a cascade of events with reversible and non-reversible outcomes. Hence, the present study was focused on conducting qualitative analysis on the effects of different light qualities on chloroplast ultrastructure, adventitious bud anatomy, and genetic fidelity of 12 weeks old in vitro Zingiber officinale var. rubrum Theilade. The study was conducted by integrating white (400–700 nm), far-red (720 nm), blue (460 nm), green (530 nm), red (660 nm), and dichromatic purple (400–660 nm) light-emitting diodes (LEDs). Chloroplasts under white, blue, and purple LEDs showed well-developed grana thylakoid and stroma thylakoid. Far-red, green, and red LEDs distorted chloroplast ultrastructure with an accumulation of starch grains. LED mediated spectral quality customised within the visible spectrum retained meristematic characteristics, vascular initiation, and oleoresin accumulation in the adventitious bud. Furthermore, clonal fidelity assessed using direct amplification of minisatellite DNA regions (DAMD) markers revealed genetic similarities of more than 99% for plantlets regenerated under LED irradiation. The results demonstrated that micropropagation systems integrated with LED spectral quality alters plant chloroplast ultrastructure by the effects on starch accumulation but produced normal adventitious bud phenology. A high genetic similarity percentage suggests effective and promising application of LED spectrum to produce true to type in vitro Z. officinale var. rubrum Theilade plantlets.

Mass COVID-19 patient screening using UvsX and UvsY mediated DNA recombination and high throughput parallel sequencing

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10–20% of symptomatic patients, particularly those with underlying medical conditions. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. A number of molecular assays are currently used to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening of COVID-19 that may be modified for application to any infectious or non-infectious genome.

Direct multiplex recombinase polymerase amplification for rapid detection of S taphylococcus aureus and P seudomonas aeruginosa in food.

Food and beverage poisoning is detrimental to people's health since it can lead to fever, stomachaches, and even death. To rapidly detect the presence of foodborne pathogens, conventional PCR assays are currently widely employed. Meanwhile, isothermal PCR methods, in which the amplification reactions take place at a low and constant temperature, have lately emerged as effective and alternative means for quickly identifying pathogens in low-resource settings. Staphylococcus aureus and Pseudomonas aeruginosa are two of the most concerning foodborne bacterial infections. In this work, an isothermal PCR assay based on the Recombinase Polymerase Amplification (RPA) method was developed to simultaneously detect S. aureus and P. aeruginosa with high sensitivity and specificity. The limit of detection for multiplex RPA was 10 and 30 fg/reaction of S. aureus and P. aeruginosa genomic DNA, respectively. Furthermore, the reaction time was reduced to only 25 minutes, with a low incubation temperature of 39°C. Multiplex RPA reactions, in particular, were successful in directly identifying as low as 1 and 5 CFU/reaction of S. aureus and P. aeruginosa cells, respectively, without the need for DNA genome extraction. Moreover, the multiplex RPA reliably detected the two foodborne bacteria in milk, fruit juice, and bottled water samples. In conclusion, the direct multiplex RPA reported in this work offers a quick, easy, sensitive, and effective alternative approach for detecting the presence of S. aureus and P. aeruginosa without the requirement of a pricey instrument or highly-trained personnel.

Sample-to-answer, extraction-free, real-time RT-LAMP test for SARS-CoV-2 in nasopharyngeal, nasal, and saliva samples: Implications and use for surveillance testing.

The global COVID-19 pandemic has highlighted the need for rapid, accurate and accessible nucleic acid tests to enable timely identification of infected individuals. We optimized a sample-to-answer nucleic acid test for SARS-CoV-2 that provides results in <1 hour using inexpensive and readily available reagents. The test workflow includes a simple lysis and viral inactivation protocol followed by direct isothermal amplification of viral RNA using RT-LAMP. The assay was validated using two different instruments, a portable isothermal fluorimeter and a standard thermocycler. Results of the RT-LAMP assay were compared to traditional RT-qPCR for nasopharyngeal swabs, nasal swabs, and saliva collected from a cohort of patients hospitalized due to COVID-19. For all three sample types, positive agreement with RT-LAMP performed using the isothermal fluorimeter was 100% for samples with Ct <30 and 69-91% for samples with Ct <40. Following validation, the test was successfully scaled to test the saliva of up to 400 asymptomatic individuals per day as part of the campus surveillance program at Rice University. Successful development, validation, and scaling of this sample-to-answer, extraction-free real-time RT-LAMP test for SARS-CoV-2 adds a highly adaptable tool to efforts to control the COVID-19 pandemic, and can inform test development strategies for future infectious disease threats.

Generation of Intense and Temporally Clean Pulses—Contrast Issues of High-Brightness Excimer Systems

In high-brightness excimer systems, the direct amplification of short pulses allows temporal filters to be integral parts of the ultraviolet (UV) amplifier chain, where the only origin of the noise is the amplified spontaneous emission (ASE), generated by the amplifier(s) following the filter. The ASE, however, develops faster than the short main pulse; in this paper, the dynamic short- and long-pulse amplification properties of KrF, XeCl and XeF excimers are studied, with special emphasis on the temporal contrast. It was found that, beyond the saturation of amplification, the relaxation of the B state in KrF, together with the contribution of the absorption of the transiently populated X state in XeCl and XeF, are the main limitations for both the extraction efficiency and the contrast. For all excimers, the stimulated transition rates and the dependence of the achievable contrast on the level of saturation were derived. Local quantities were introduced to characterize the deterioration of the contrast for a unit gain length of KrF amplifiers. A KrF power amplifier of limited gain (G ≈ 3), following the newly introduced nonlinear Fourier filter, is capable of reaching contrast levels beyond the previously reported 1011–1012.

Isothermal RNA Amplification for the Detection of Viable Pathogenic Bacteria to Estimate the Salmonella Virulence for Causing Enteritis.

Viable foodborne pathogens can cause intestinal infection and food poisoning. Herein, we reported an RNA assay allowing for sensitive (close to 1 CFU and 1% viable bacteria detectable) and rapid (within 2.5 h) detection of viable pathogenic bacteria by coupling isothermal RNA amplification (nucleic acid sequence-based amplification, NASBA) with a CRISPR/Cas13a system. NASBA allowed direct amplification of 16S rRNA extracted from viable S. enterica (RNAs degrade rapidly in dead bacteria), and the specificity of amplification was ensured using Cas13a/crRNA to recognize the amplicons. We used the CRISPR/Cas13-based NASBA assay (termed cNASBA assay) to investigate the in vivo colonization and intestinal infection of S. enterica in mice. We found that S. enterica was mainly colonized at the cecum, colon, and rectum, and the severity of enteritis caused by S. enterica was determined by the number of viable S. enterica rather than the total count of S. enterica. The cNASBA assay can quantify viable S. enterica and thus can improve the accuracy of virulence estimation compared to qPCR. It shows promise as a reliable tool for monitoring pathogen contamination and biosafety control.

A single direct amplification method for forensic casework references on a variety of substrates

A single direct amplification method for buccal and blood reference samples, on different substrates, containing a wide range of cellular material, was developed. This method uses the GlobalFiler™ STR kit, which was designed for amplification of previously purified and quantified DNA. Buccal samples on cotton swabs, Bode collectors, and Whatman® FTA® cards, and liquid blood, blood on Whatman® 903 and FTA® cards, and autopsy bloods were examined. A first pass success rate, that is, complete concordant profiles with no GeneMapper™ ID-X quality flags, of > 95% was obtained. This method involves the lysis of reference samples (tips of cotton swabs, double 4.8 mm diameter Bode or FTA® buccal punches, and single 4.8 mm diameter blood punches on Whatman® 903 or FTA® cards) in 100 µl of Prep-n-Go™ lysis buffer, and amplification of 2 µl of the lysate with 25 cycles. Data collection is optimized by use of the spectral pull-up reduction and off-scale recovery features of the AB3500 Genetic Analyzer Data Collection version 4 software, and by injection of PCR products for two different lengths of time (15 and 7 s). Data was analyzed with GeneMapper™ ID-X version 1.6, which allowed an increased signal dynamic range (maximum peak height of 64000 RFU), and by employing a 10% global filter, and a 20% global stutter filter. Complete GlobalFiler™ profiles were generated from as little as 5 µl of blood, 1 buccal swipe on cotton swab and Bode collectors, and collection of buccal cells deposited on the foam heads of EasiCollect™ FTA® collectors for 5 s.

Phenotype\u2013genotype correlation in patients with typical and atypical branchio-oto-renal syndrome

Some patients have an atypical form of branchio-oto-renal (BOR) syndrome, which does not satisfy the diagnostic criteria, despite carrying a pathogenic variant (P variant) or a likely pathogenic variant (LP variant) of a causative gene. P/LP variants phenotypic indices have yet to be determined in patients with typical and atypical BOR syndrome. We hypothesized that determining phenotypic and genetic differences between patients with typical and atypical BOR syndrome could inform such indices. Subjects were selected from among patients who underwent genetic testing to identify the cause of hearing loss. Patients were considered atypical when they had two major BOR diagnostic criteria, or two major criteria and one minor criterion; 22 typical and 16 atypical patients from 35 families were included. Genetic analysis of EYA1, SIX1, and SIX5 was conducted by direct sequencing and multiplex ligation-dependent probe amplification. EYA1 P/LP variants were detected in 25% and 86% of atypical and typical patients, respectively. Four EYA1 P/LP variants were novel. Branchial anomaly, inner ear anomaly, and mixed hearing loss were correlated with P/LP variants. Development of refined diagnostic criteria and phenotypic indices for atypical BOR syndrome will assist in effective detection of patients with P/LP variants among those with suspected BOR syndrome.

Fully soft organic electrochemical transistor enabling direct skin-mountable electrophysiological signal amplification.

Here, we propose fully soft OECTs with all soft components, including a PEDOT:PSS-based soft channel, which show substantial mechanical/electrical properties. In addition, the further demonstrated skin-mountable amplifier implies the strong potential of this work to be an innovative development in wearable electronics.

Faradaic-free electrokinetic nucleic acid amplification (E-NAAMP) using localized on-chip high frequency Joule heating.

We present a novel Faradaic reaction-free nucleic acid amplification (NAA) method for use with microscale liquid samples. Unlike previous Joule heating methods where the electrodes produce electrolysis gaseous by-products and require both the electrodes be isolated from a sample and the venting of produced electrolysis gas, our electrokinetic Nucleic Acid Amplification (E-NAAMP) method alleviates these issues using a radio frequency (RF) alternating current electric field. In this approach, a pair of microscale thin film gold electrodes are placed directly in contact with a nucleic acid reaction mixture. A high frequency (10-40 MHz) RF potential is then applied across the electrode pair to induce a local Ohmic current within the sample and drive the sample temperature to increase by Joule heating. The temperature increase is sustainable in that it can be generated for several hours of constant use without generating any pH change to the buffer or any microscopically observable gaseous electrolysis by-products. Using this RF Joule heating approach, we demonstrate successful direct thermal amplification using two popular NAA biochemical reactions: loop-mediated isothermal amplification and polymerase chain reaction. Our results demonstrate that a simple microscale electrode structure can be used for thermal regulation for NAA reactions without observable electrolytic reactions, minimal enzyme activity loss and sustained (>50 h use per device) continuous operations without electrode delamination. As such, E-NAAMP offers substantial miniaturization of the heating elements for use in microfluidic or miniaturized NAA reaction systems.

Simple and Rapid Detection of ESBL blaSHV gene from an Urban River in Tokyo by Loop-Mediated Isothermal Amplification.

Extended-spectrum β-lactamases (ESBLs) are produced mainly by gram-negative bacteria in Enterobacteriaceae. One of the major types of ESBLs is sulfhydryl variable (SHV) -type ESBL. Herein, we attempted to develop a simple and rapid method for the detection of the ESBL blaSHV gene by loop-mediated isothermal amplification (LAMP) . The five-primer set designed could amplify blaSHV gene at an isothermal temperature of 65℃. The detection limit of the LAMP method with the LF loop primer was 1 copy/tube, which was 10,000-fold more sensitive than that of the conventional PCR. The LAMP assay could also detect the direct amplification of blaSHV gene from a single river water sample in Tokyo. The LAMP method has great potential for applications in hospital, soil and water environment, food, and livestock.

Exploring tapelifts as a method for dual workflow STR amplification

Although a version of direct PCR is implemented in forensic laboratories for reference material, its incorporation into workflow for the analysis of touch DNA, as a form of latent DNA, from casework exhibits is not. In addition to concerns about increased sensitivity causing more complex mixtures or the generation of more genetic data implicating an individual superfluous to the context of the alleged event, the complete use of the collected sample in the PCR as template has meant that there is no possibility for data reproducibility when needed. Here it is proposed that the use of tapelifts in touch DNA collection can facilitate replicate direct PCR analysis from a single sample allowing the sample to be re-tested. If all portions of the tapelift result in profiles with allelic and likelihood ratio concordance, these sub-samples may be accepted as technical replicates, thus meeting any accreditation guideline requirements. Furthermore, we assess the use of a single tapelift for both direct PCR and extraction-based PCR workflows to illustrate the potential for benefits of both systems to be facilitated. DNA was deposited by three donors onto six substrates with five sample replicates of each condition. Separation of each tapelift into three portions for three direct PCRs ensued using VeriFiler™ Plus. Separation of single tapelifts into three direct PCRs showed no statistical difference in donor allele calls or RFU, or subsequent LRs associated with their profiles. Comparison of profiles within the single tapelift showed more similarity, with high mixture-to-mixture match likelihoods, than when these sub-samples were compared with profiles generated from other samples. This allows each sub-sample taken from the tapelift to be considered as technical replicates. For dual workflow facilitation assessment, one donor deposited DNA through touch onto six substrates with five research replicates of each. Separation of single tapelifts into two portions, one for direct PCR and the retention and use of the remaining portion for extraction and subsequent PCR, showed no significant difference in allelic yield and subsequent donor comparison LRs. Comparison of deconvoluted profiles produced from a single tapelift showed high mixture-to-mixture match likelihoods, supporting DNA donor concordance. This indicates that removing a portion of a tapelift for direct PCR amplification, while processing the remainder through standard processes, allows increased sensitivity through direct PCR while offering the preparation of an eluate suitable for repeated analyses.

Improved direct lysis PCR amplification method of microalgal culture for sequencing and species identification

Molecular approach plays important role in species identification for microalgae which involves sequencing of specific DNA barcode present in the genome. This approach involved preparation of template DNA for polymerase chain reaction (PCR) which is time consuming and requires large amounts of algal cells. Microalgal direct PCR have been used frequently for species identification, which simplified the DNA isolation procedure. However, the recent attempts to amplify the rbcL gene of microalga using the previously reported protocol led to poor repeatability. In this study, Nannochloropsis gaditana NIES-2587 was cultured in f/2 liquid medium. The culture growth was estimated on optical density value and the lysis process was improved using gradual temperature procedure during the PCR process. The same culture was extracted using manual DNA extraction method for comparison. The DNA obtained from both methods were amplified using RbclN primer pair to amplify 1486 bp partial sequence of Nannochloropsis rbcL gene, followed by the sequencing of the PCR product. Molecular identification based on the sequence result and BLAST analysis indicated that direct PCR and manual DNA extraction methods successfully produced high sequences result and confirmed the identity of microalgae species into N. gaditana strain CCMP527 with a genetic similarity of >99%.

Large mode area double-clad ytterbium-doped spun tapered fiber

We performed a comparative study of Yb-doped double-clad large mode area ( M F D = 31 µ m ) spun tapered fibers with different pitch lengths. A novel type of spun tapered fibers with constant pitch lengths of 7.5, 15, and 30 mm, internal birefringence as low as ∼ 10 − 8 , and perfect output beam quality ( M 2 &lt; 1.2 ) has been manufactured and investigated. We demonstrated the efficient direct amplification of a picosecond pulsed signal (95 ps/100 MHz, 1064 nm) in the spun active tapered fiber with optimized geometry providing an average output power of 71 W (gain 32 dB, 74% slope efficiency). Efficient green light generation ( λ = 532 n m ) with 17 W average power (1.7 µJ) and 80 pm linewidth was demonstrated using the developed master oscillator power amplifier system.

Functional hydrogel for fast, precise and inhibition-free point-of-care bacteria analysis in crude food samples

In this work, instead of performing nucleic acid amplification in the bulk solution, we report a nanoporous hydrogel with controlled release function for rapid, precise, and inhibition-free nucleic acid analysis in crude food samples. The cross-linked PEG hydrogel with nanoporous structures possesses adsorption, release, separation, restriction and self-cleaning abilities. When digital loop-mediated isothermal amplification (LAMP) was performed inside this hydrogel, the surrounding nanostructure act as a temporary reservoir for reagents storage and release them on demand during or after amplification. Meanwhile, the restricted nanoconfined environment of hydrogel also favor the enzymatic amplification process. Thus, an enhanced signal readout, robust anti-inhibition, faster amplification rate, more products yields and specific amplification without primer-dimers were obtained. Moreover, direct amplification in untreated complex food sample was successfully performed inside hydrogel without any sample pretreatment, while conventional droplets digital LAMP failed for detection. Absolute quantification of Escherichia coli and Salmonella typhi directly in fresh fruit and vegetables was achieved within 20 min, with high precision and sensitivity down to single cell. This novel lab-on-hydrogel concept has an enormous potential for future molecular diagnostic assays, and can be also applied for other point-of-care assays.

Development and Forensic Application of EX20+30Y Kit.

To develop a multiplex PCR amplification system (EX20+30Y for short) of 19 autosomes, 30 Y-STR loci plus the gender indicator, and evaluate its forensic application value. With the six-color fluorescence labeling technology, a multiplex amplification system of 19 autosomal STR loci and 30 Y-STR loci plus the gender indicator was constructed. Blood samples from 210 unrelated individuals, 69 daily case samples and standard samples 9948 and 9947A were collected for loci detection and analysis. The EX20+30Y multiplex amplification system was evaluated by its sensitivity, mixed sample detection ability, species specificity, balance, direct amplification ability, sample applicability and anti-inhibition ability. Multiplex amplification of blood samples from 210 unrelated individuals by the detection system obtained accurate genotyping results. The detection sensitivity of standard samples was 0.125 ng and the species specificity was high. The 69 samples from daily cases were genotyped correctly. Moreover, standard sample 9948 could be accurately genotyped even if the samples contained a certain concentration of inhibitors. The multiplex amplification system established in this study can conduct combined examination of 19 autosomes, 30 Y-STR loci plus the gender indicator with accurate genotyping and high sensitivity. It has a good forensic application prospect.