Standard Real-time PCR Research Articles

Microsphere integrated convective amplification sensor for all-in-one-pot detection of Mycoplasma pneumoniae

The integration of isothermal amplification with CRISPR-Cas systems play a crucial role in the point-of-care test of infectious pathogens, owing to their specificity, sensitivity, and portability. However, these systems lack rigorous internal amplification control for monitoring false negatives and rely on carefully designed guide sequences, which barriers to their further promotion and application. Additionally, the complexity of sample purification and the laborious preparation of liquid reagents significantly limit these systems to ready-to-use and direct sample-to-answer detection in on-site diagnosis. Here, we combined ready-to-use lyophilized microsphere, thermal lysis of pathogens, guide-sequence-independent convective amplification, internal amplification control for monitoring negative false and a portable multi-channel instrument to develop a microsphere integrated convective amplification sensor (MICASOR) system for on-site detection. MICASOR integrated the overall workflow of DNA testing into one-pot to achieve all-in-one-pot detection for on-site diagnosis. MICASOR is a rapid, potable, ready-to-use and guide-sequence-independent platform and can monitor false negatives. MICASOR can detect as low as 1 copy/μL of clinical samples of Mycoplasma pneumoniae within 15 minutes. Validated by 30 real clinical samples, MICASOR showed 100 % agreement with gold standard real-time PCR. These results suggest that MICASOR has great potential to promote the control of infectious diseases in the outpatients, communities and poor resource areas.

Development and validation of a droplet digital PCR assay for Nipah virus quantitation

BackgroundNipah virus (NiV) is a zoonotic pathogen that poses a significant threat because of its wide host range, multiple transmission modes, high transmissibility, and high mortality rates, affecting both human health and animal husbandry. In this study, we developed a one-step reverse transcription droplet digital PCR (RT-ddPCR) assay that targets the N gene of NiV.ResultsOur RT-ddPCR assay exhibited remarkable sensitivity, with a lower limit of detection of 6.91 copies/reaction. Importantly, it displayed no cross-reactivity with the other 13 common viruses and consistently delivered reliable results with a coefficient of variation below 10% across different concentrations. To validate the effectiveness of our RT-ddPCR assay, we detected 75 NiV armored RNA virus samples, mimicking real-world conditions, and negative control samples, and the RT-ddPCR results perfectly matched the simulated results. Furthermore, compared with a standard quantitative real-time PCR (qPCR) assay, our RT-ddPCR assay demonstrated greater stability when handling complex matrices with low viral loads.ConclusionsThese findings show that our NiV RT-ddPCR assay is exceptionally sensitive and provides a robust tool for quantitatively detecting NiV, particularly in stimulated field samples with low viral loads or complex matrices. This advancement has significant implications for early NiV monitoring, safeguarding human health and safety, and advancing animal husbandry practices.

Performance evaluation of loop-mediated isothermal amplification, polymerase chain reaction and real-time polymerase chain reaction methods to detect Neisseria gonorrhoeae among symptomatic patients from India.

Background: Neisseria gonorrhoeae is one of the most important causative organisms in causing sexually transmitted infections. The clinical presentation of gonorrhoea mimics the symptoms of other sexually transmitted infections, and a proper diagnosis of the same is therefore crucial in patient management. The current study intended to compare different in-house molecular methods: that is, conventional PCR, real-time PCR, and LAMP assay for detection of N. gonorrhoeae. Methods: A total of 163 samples were collected from 145 patients who presented with urethral and vaginal discharge. Collected samples were processed for culture on GC agar base, and three different molecular diagnostic tests (conventional PCR, real-time PCR, and LAMP assay) were performed simultaneously on all the samples. Results: Culture of N. gonorrhoeae was positive in 17 out of 21 (80.9%) swab samples. With culture as the gold standard method, conventional and real-time PCR had a sensitivity of 94.1%, whereas the sensitivity of the LAMP assay was found to be 88.2%. All three methods had a specificity of 100%. In addition to swab samples, evaluation of urine samples by different molecular methods yielded a good concordance with a kappa value of 0.85 by conventional PCR and real-time PCR showing a perfect level of agreement, while the LAMP assay was found to have a substantial level of agreement. Conclusion: LAMP assay had a comparable diagnostic accuracy to other molecular methods for the detection of N. gonorrhoeae and can be used as a point-of-care test in resource-limited settings.

Real-time PCR detection of PI*S and PI*Z alleles of SERPINA1 gene using SYBR green

BackgroundAlpha-1 antitrypsin deficiency is an underdiagnosed genetic condition that predisposes to pulmonary complications and is mainly caused by rs28929474 (PI*Z allele) and rs17580 (PI*S allele) mutations in the SERPINA1 gene. ObjectiveDevelopment of a homogeneous genotyping test for detection of PI*S and PI*Z alleles based on the principles of allele-specific PCR and amplicon melting analysis with a fluorescent dye. MethodsSixty individuals, which included all possible genotypes that result from combinations of rs28929474 and rs17580 single nucleotide variants, were assayed with tailed allele-specific primers and SYBR Green dye in a real-time PCR machine. ResultsA clear discrimination of mutant and wild-type variants was achieved in the genetic loci that define PI*S and PI*Z alleles. Specific amplicons showed a difference of 2.0 °C in melting temperature for non-S and S variants and of 2.9 °C for non-Z and Z variants. ConclusionsThe developed genotyping method is robust, fast, and easily scalable on a standard real-time PCR platform. While it overcomes the handicaps of non-homogeneous approaches, it greatly reduces genotyping costs compared with other homogeneous approaches.

An instrument-free, integrated micro-platform for rapid and multiplexed detection of dairy adulteration in resource-limited environments

In dairy industry, expensive yak's milk, camel's milk, and other specialty dairy products are often adulterated with low-cost cow's milk, goat's milk and so on. Currently, the detection of specialty dairy products typically requires laboratory settings and relies on skilled operators. Therefore, there is an urgent need to develop a multi-detection technology and on-site rapid detection technique to enhance the efficiency and accuracy of the detection of specialty dairy products. In this study, we introduced a fully integrated and portable microfluidic detection platform called Sector Self-Driving Microfluidics (SDM), designed to simultaneously detect eight common species-specific components in milk. SDM integrated nucleic acid extraction, purification, loop-mediated isothermal amplification (LAMP), and lateral flow strip (LFS) detection functions into a closed microfluidic system, enabling contamination-free visual detection. The SDM platform used a constant-temperature heating plate, powered by a mobile battery, eliminated the need for additional power support. The SDM platform achieved nucleic acid enrichment and transfer through magnetic force and liquid flow driven by capillary forces, operating without external pumps. The standalone SDM platform could detect dairy components with as low as 1% content within 1 h. Validation with 35 commercially available samples demonstrated 100% specificity and accuracy compared to the gold standard real-time PCR. The SDM platform provided the dairy industry with an efficient, convenient, and accurate detection tool, enabling rapid on-site testing at production facilities or sales points. This facilitated real-time monitoring of quality issues during the production process, quickly identifying potential risks and preventing substandard products from entering the market.

Reduced SIRT1 and SIRT3 and Lower Antioxidant Capacity of Seminal Plasma Is Associated with Shorter Sperm Telomere Length in Oligospermic Men.

Infertility affects millions of couples worldwide and has a profound impact not only on their families, but also on communities. Telomere attrition has been associated with infertility, DNA damage and fragmentation. Oxidative stress has been shown to affect sperm DNA integrity and telomere length. Sirtuins such as SIRT1 and SIRT3 are involved in aging and oxidative stress response. The aim of the present study is to determine the role of SIRT1 and SIRT3 in regulating oxidative stress, telomere shortening, and their association with oligospermia. Therefore, we assessed the protein levels of SIRT1 and SIRT3, total antioxidant capacity (TAC), superoxide dismutase (SOD), malondialdehyde (MDA) and catalase activity (CAT) in the seminal plasma of 272 patients with oligospermia and 251 fertile men. We also measured sperm telomere length (STL) and leukocyte telomere length (LTL) using a standard real-time quantitative PCR assay. Sperm chromatin and protamine deficiency were also measured as per standard methods. Our results for oligospermic patients demonstrate significant reductions in semen parameters, shorter STL and LTL, lower levels of SOD, TAC, CAT, SIRT1 and SIRT3 levels, and also significant protamine deficiency and higher levels of MDA and DNA fragmentation. We conclude that a shorter TL in sperms and leukocytes is associated with increased oxidative stress that also accounts for high levels of DNA fragmentation in sperms. Our results support the hypothesis that various sperm parameters in the state of oligospermia are associated with or caused by reduced levels of SIRT1 and SIRT3 proteins.

Manually pressurized droplet digital PCR chip for rapid SARS-CoV-2 diagnostics.

Droplet digital PCR (ddPCR) is a technique in which PCR reaction is divided into thousands of nanoliter-sized droplets and has proven to be a great tool in virus diagnostics. Compared to the gold standard system quantitative real-time PCR (RT-qPCR), ddPCR functions particularly well when dealing with samples with low template counts, such as viral concentration. This feature makes the technique suitable for early detection of the virus. In this study, a novel portable PDMS ddPCR chip is introduced. The chip functions without external pumps using manual pressurization with a multichannel pipet. The created droplets are monodispersed and form a monolayer on the chip's collection chamber, from where they can be effortlessly imaged. Droplets were analyzed and counted using artificial intelligence. The use of the manually pressurized chip was demonstrated for a SARS-CoV-2 assay, which takes advantage of isothermal strand invasion-based amplification (SIBA) technology, allowing quick and accurate, even point-of-care analysis of the sample. The results demonstrate that SIBA assays can be divided into nanoliter-sized droplets and used as quantitative assays, giving an approximation of the samples' viral count.

Evaluation of Metabarcoding Methods for Plant Disease Surveillance

Metabarcoding holds great potential for general plant pathogen surveillance by providing an untargeted profile of the host microbiome. However, the standard marker utilized for microbiome analysis of prokaryotes, the 16 rRNA gene, offers limited diagnostic utility as it provides phylogenetic resolution primarily at the genus level, and universal primers often co-amplify plant DNA. Here, we evaluated two recently published universal primer sets targeting the DNA gyrase and RNA polymerase ß subunit genes ( gyrB and rpoB, respectively), relative to a plant discriminating 16S primer set (799F/1115R), as disease surveillance tools. Comparative analysis of a mock bacterial community, as well as naturally infected citrus variegated chlorosis samples, indicated that the gyrB method displayed optimal performance in the amplification of DNA across a broad taxonomic spectrum of plant-pathogenic bacteria, providing resolution at the species and, in some cases, subspecies levels. It also generated high-quality datasets with minimal to no co-amplification of plant DNA, outperforming the rpoB and 16S assays in these regards. Further evaluation revealed that the gyrB method displayed an overall linear trend in the detection of several diverse bacterial pathogens; however, it was at least an order of magnitude less sensitive than a standard real-time PCR assay. Finally, we demonstrate the potential of this method to disentangle a mixed population of Pantoea spp. associated with a rice-bacterial blight outbreak and identify putative novel pathogens that would otherwise be overlooked with conventional PCR-based tests. This work represents steps toward establishing a robust, untargeted metabarcoding method for general plant disease surveillance. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Effect of breastfeeding on the HMGA2 single nucleotide polymorphisms in relation to first primary tooth emergence among Iraqi infants

Abstract Backgrounds: Breastfeeding provides a large group of functional components that improve the infant’s health and development. additionally, the impact of human milk and genetic variants on complicated outcomes has also been the subject of several gene–nutrition interaction studies. This could also play a key part in the kinship that exists between offspring and their parents. Objectives: The purpose of this study was to compare breastfeeding to infants who were fed formula in order to determine if human milk affected the timing of teething in relation to genetic polymorphisms. Materials and Methods: This study is a cross-sectional comparative study concerning genetic analysis with a prospective view concerning the timing of eruption. The chosen sample included 40 breastfed infants and 40 infants who consumed formula milk. It was compiled from Baghdad’s health centers. For the genetic analysis, DNA samples were obtained from saliva throughout the buccal swab. Standard DNA extraction and real-time PCR techniques have been used according to the manufacturer’s instructions for the Quick-DNA™ MiniPrep kit (catalog nos. D3024 and D3025). For the whole sample, clinical examination and monitoring for emerging teeth were performed using a particular criterion. Results: findings showed that there were highly significant differences between breastfeeding and early eruption time for the first primary tooth. Also, there was a significant association between single nucleotide polymorphisms in the heterozygous AG genotype and eruption time. Conclusion: The results of the current study may support the genetic influence of breast milk on early primary teeth eruption when juxtaposed with infants fed formula.

B-241 A Novel Approach to Develop Multiplex Loop Mediated Amplification (LAMP) Assays

Abstract Background Loop mediated amplification (LAMP) technology is a sensitive, accurate and rapid method for point-of-care (POC) molecular diagnostic detection. One disadvantage is that current LAMP methods can detect only one target per reaction whereas in a standard real-time PCR assay, multiple targets (3–4) can be detected in a single reaction. This is because current methods of detection for amplification products in a LAMP assay are not sequence-specific. Common detection methods include turbidity, use of color-changing dye or intercalating dye in the reaction mix, gel electrophoresis, etc. These methods are not sequence-specific and, thus, unable to differentiate between targets if amplified in a single tube, with the exception of lateral flow devices or melt analysis, which add time, complexity, and cost. To overcome this challenge, we have developed a novel method which simplifies development of multiplex LAMP without use of any additional primers/probes/reagents or steps. Methods Triplex RT-LAMP assay was developed for rapid detection of foot-and-mouth disease virus (FMDV) at POC. For this, LAMP primers targeting two different regions of 3D pol gene of FMDV were designed. For use as internal control (IC), LAMP primers were also designed targeting a conserved region of the bovine 18S rRNA gene. For multiplexing, best performing primer sets for each target were labelled with a unique fluorophore at 5′ end and a quencher (BHQ-1) at 3′ end. FMDV-1 primers were labeled with Cy5 fluorophore, FMDV-2 primers were labelled with Texas Red (TxR) fluorophore, and 18S rRNA with FAM fluorophore. Triplex RT-LAMP assay was performed using a specially formulated Multiplex Isothermal Master Mix but without any intercalating dye added to the mix. All 3 primer mixes were added to the Master Mix and reactions were carried out in a real-time thermocycler at 72°C for 30 min, with data collected every 30 seconds. Reaction kinetics were monitored as an increase in fluorescence associated with the accumulation of double-stranded DNA in respective channels (FAM for 18S rRNA, Cy5 for FMDV-1, and TxR for FMDV-2). Assay sensitivity was determined by testing 10-fold serial dilution of respective template, individually as well as mixed together. Results Results obtained showed feasibility of triplex RT-LAMP assay for simultaneous detection of 3 targets in a single reaction. Sensitivity and specificity of triplex RT-LAMP was comparable to control reactions (RT-LAMP assay with intercalating dye) for each target. No cross reactivity or non-specific amplification was observed with any of the primer designs. Conclusions This newly developed method allows detection of up to 3 targets in a single reaction without affecting the assay performance. This method is much easier to develop and optimize than previously reported methods as it uses standard LAMP primers without need of any additional primers/probes.

Unamplified system for sensitive and typing detection of ASFV by the cascade platform that CRISPR-Cas12a combined with graphene field-effect transistor

At present, the 100% case fatality and the cross-infection of virus strains make the ASFV 's harm to society continue to expand. The absence of an effective commercial vaccine poses early detection remains the most effective means of curbing ASFV infection. Here, we report a cascaded detection platform based on the CRISPR-Cas12a system combined with graphene field-effect transistor sensors. The cascade platform could detect ASFV as low as 0.5 aM within 30 min and achieve typing of wild and vaccine strains of ASFV in a single detection system. The evaluation of 16 clinical samples proved that, compared with the gold standard Real-time PCR method, this platform has outstanding advantages in sensitivity, specificity and typing. Combining CRISPR-Cas12a's high specificity with the bipolar electric field effect of graphene field-effect transistor, the cascade platform is expected to achieve clinical application in the field of DNA disease detection, and provides a new direction for multi-strain disease typing.

Diagnostic Agreement of SARS-CoV-2 Lateral Flow Antigen Assay with the Cycle Threshold Values of RT-PCR

COVID-19 detection via lateral flow antigen assays (LFA) are rapid and economically acquiescent to infrastructure facile healthcare settings. Early, prompt identification of cases to facilitate patient isolation and supportive management is the essence of rapid diagnostic tests. Given the backdrop of post COVID-19 pandemic-molecular testing still remains a costly affair. Additionally, molecular assays are incapable of distinguishing remnant RNA from replication competent viruses. In this scenario, we explore the diagnostic consonance of SARS-CoV-2 LFAs with RT-PCR cycle threshold, in a likelihood that it could be used as a surrogate marker for infection transmissibility. Rapid COVID-19 LFA results were compared with Real-time PCR for detection of SARS-CoV-2 in nasopharyngeal swabs. Two hundred rapid antigen positive nasopharyngeal swabs obtained from COVID-19 suspects/contacts/preoperative/screening patients were subjected to RT-PCR to study the correlation with cycle threshold (CT) values obtained for all the antigen positive cases. 200 Rapid COVID-19 LFA positive samples were analyzed in the present study. Amidst the LFA positive samples included in the study 187 (93.5%) were found to have concordant results when subjected to the gold standard Real-time PCR. Discordant results were documented in 13 (6.5%) COVID-19 LFA positive samples which were found to be negative by RT-PCR. The average Cycle threshold values were found to be 23.75 for E gene, 25.36 for N gene and 24.07 for RdRp gene. The average PCR Cycle threshold of LFA positive cases remained significantly undeterred (p<0.5) throughout the time period of the study stipulating the undaunted viral load across the different waves of the pandemic. Maximum association of LFA positivity with symptom-manifestation was seen during the 1st wave of COVID-19 (September-December 2020 in India). The association of symptoms with LFA test positivity reduced to a significant extent during the 3rd wave of the pandemic in January 2022 (p<0.5) indicating the reduced clinical severity but not infectivity of the SARS-CoV-2 infection during the 3rd wave of the pandemic. Lateral flow assay based diagnostic tests are technically & economically convenient modalities with significant interest concordance in comparison with RT-PCR. Definitive advantage in terms of achieving quick patient triage and thereby patient management can be achieved with the use of these tests.

Identification of horse, donkey and pig ingredients by species-specific ERA-based methods to assess the authenticity of meat products

Ensuring the authenticity of horse-, donkey- and pig-related meat products is of great religious and economic significance. In this study, with the mitochondrial genes of ATPase 6 or ND2 as targets, the species-specific real-time enzymatic recombinase amplification (real-time ERA) and ERA combined with lateral flow strip (LFS ERA) assays for horse, donkey and pig ingredients were developed. The optimization results showed that both ERA assays had the same optimal temperature for the detection of horse, donkey and pig ingredients, i.e., 39 °C for real-time ERA and 37 °C for LFS ERA, respectively. The assays could be performed simultaneously and the results could obtained within 25 min. Both developed assays showed good specificity and no cross-reactions with other common meat species were observed. The limit of detection of the developed ERA assays for horse, donkey and pig ingredients was 10 pg genomic DNA/per reaction and 0.1% target meat (w/w). Their applicability were verified with 45 donkey meat products (both raw and marinated minced meat), and the results showed that the detection rate of horse or pig ingredient was as high as 53.33%, including a complete substitution or partial substitution of donkey meat. The results were consistent with those of the standard real-time PCR in China, but the whole detection time was significantly reduced from 2 h to 35 min. It is concluded that the developed real-time ERA and LFS ERA assays are feasible for the rapid authentication of horse, donkey and pig meats and identification of the adulteration of donkey meat-related products.

Detection of BRAF mutations in malignant melanoma and colorectal cancer by SensiScreen® FFPE BRAF qPCR assay.

Mutations in BRAF exon 15 lead to conformational changes in its activation loops, resulting in constitutively active BRAF proteins which are implicated in the development of several human cancer types. Different BRAF inhibitors have been developed and introduced in clinical practice. Identification of BRAF mutations influences the clinical evaluation, treatment, progression and for that reason a sensitive and specific identification of BRAF mutations is on request from the clinic. Here we present the SensiScreen® FFPE BRAF qPCR Assay that uses a novel real-time PCR-based method for BRAF mutation detection based on PentaBases proprietary DNA analogue technology designed to work on standard real-time PCR instruments. The SensiScreen® FFPE BRAF qPCR Assay displays high sensitivity, specificity, fast and easy-to-use. The SensiScreen® FFPE BRAF qPCR Assay was validated on two different FFPE tumour biopsy cohorts, one cohort included malignant melanoma patients previously analyzed by the Cobas® 4800 BRAF V600 Mutation Test, and one cohort from colorectal cancer patients previously analyzed by mutant-enriched PCR and direct sequencing. All BRAF mutant malignant melanoma patients were confirmed with the SensiScreen® FFPE BRAF qPCR Assay and additional four new mutations in the malignant melanoma cohort were identified. All the previously identified BRAF mutations in the colorectal cancer patients were confirmed, and additional three new mutations not identified with direct sequencing were detected. Also, one new BRAF mutation not previously identified with ME-PCR was found. Furthermore, the SensiScreen® FFPE BRAF qPCR Assay identified the specific change in the amino acid. The SensiScreen® FFPE BRAF qPCR Assay will contribute to a more specific, time and cost saving approach to better identify and characterize mutations in patients affected by cancer, and consequently permits a better BRAF characterization that is fundamental for therapy decision.

Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis

Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.

Cultivable microbiota of Proteus anguinus from underground habitats and animals accidently washed to the surface in Croatia

Proteus anguinus (olm) is an amphibian endemic to underground cave systems of the Dinaric karst of Central Europe, adapted to a life in complete darkness. Recent studies have demonstrated the global decline of amphibians due to poorly understood microbiological diseases, emphasizing just how little is known about the natural microflora of amphibians. Not much is known about the olm’s microbiota in their cave habitats, and nothing is known about the microbiology of animals washed to the surface. This observational study describes the microbiological analysis of the skin, cloaca and oral cavity of six olms collected from their cave habitats, and 16 collected after being washed out by flows from two springs, to learn more about the olm’s normal microbiota and possible changes after contact with the different environmental conditions on the surface. Standard microbiological procedures, MALDI-TOF and Real-Time PCR were used for microbiological species identification. All animals tested negative for Ranavirus, Batrachochytrium dendrobatidis, Batrachochytrium salamandrivorans and Chlamydia spp. The most abundant fungi isolated were Penicillium spp. and Cladosporium spp. The washed out animals showed a higher diversity of bacterial flora than those from cave habitats, with Acinetobacter johnsonii, Aeromonas hydrophila, Bacillus spp. and Janthinobacterium lividum as the most frequently identified isolates.

Nanoparticle-Based Visual Detection of Amplified DNA for Diagnosis of Hepatitis C Virus.

Rapid, simple, and inexpensive diagnostic point-of-care tests (POCTs) are essential for controlling infectious diseases in resource-limited settings. In this study, we developed a new detection system based on nanoparticle–DNA aggregation (STat aggregation of tagged DNA, STAT-DNA) to yield a visual change that can be easily detected by the naked eye. This simplified optical detection system was applied to detect hepatitis C virus (HCV). Reverse transcription-polymerase chain reaction (RT-PCR) was performed using primers labeled with biotin and digoxigenin. Streptavidin-coated magnetic particles (1 μm) and anti-digoxigenin antibody-coated polystyrene particles (250–350 nm) were added to form aggregates. The limit of detection (LoD) and analytical specificity were analyzed. The STAT-DNA results were compared with those of the standard real-time PCR assay using serum samples from 54 patients with hepatitis C. We achieved visualization of amplified DNA with the naked eye by adding nanoparticles to the PCR mixture without employing centrifugal force, probe addition, incubation, or dilution. The LoD of STAT-DNA was at least 101 IU/mL. STAT-DNA did not show cross-reactivity with eight viral pathogens. The detection using STAT-DNA was consistent with that using standard real-time PCR.

Abstract 4104: Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology

Abstract Introduction: The FDA has approved 11 therapies for non-small cell lung cancer (NSCLC) patients diagnosed with gene fusions. Importantly, these life-saving targeted therapies remain underutilized due to a lack of cost-effective, comprehensive, and simple-to-use technologies for gene fusion detection. Our previous work using ASPYRE technology (allele-specific pyrophosphorolysis reaction; Silva et al, 2020) demonstrated detection of somatic variants at single-molecule level from DNA extracted from tumor tissue. However, DNA-based fusion detection requires tiling across large introns, which limits sensitivity particularly if breakpoints are located in repetitive regions of the genome. Here, we show that ASPYRE technology can also be utilized for highly sensitive detection of RNA fusions, scaling to over 30 fusion targets covering the main 3’ fusion partners (ALK, ROS1, RET, NTRK1, NTRK3) and from a single reverse transcription PCR (RT-PCR) amplification reaction. Methods: The ASPYRE assay consists of four sequential enzymatic stages: RT-PCR; enzymatic clean-up; combined exonuclease digestion, hybridization, pyrophosphorolysis and ligation; rolling circle isothermal amplification and detection. All targets are detected across two reaction wells using four channels on a standard real-time PCR instrument. A panel of synthetic RNA oligonucleotides comprising gene fusion boundaries was spiked into a background pool of lung-RNA, quantified using digital PCR (dPCR) and compared against a commercial reference standard to validate its suitability for assessing assay performance. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) lung tissue from healthy donors to determine assay specificity, and from FFPE lung tissue from NSCLC patients with known fusions to exemplify end-to-end sample analysis. Results: We demonstrate equivalence of our in-house reference samples to commercial reference standards. Using serial dilutions of input RNA standards, we show that detection by ASPYRE is consistent with detection of single molecules as input for common ROS1, ALK, RET and NTRK3 fusions. In addition, ASPYRE was able to detect previously confirmed ROS1 and ALK fusions and MET exon skipping variants in FFPE samples. ASPYRE had high specificity, correctly confirming the absence of RNA fusions and exon skipping variants in healthy control FFPE samples. Conclusions: ASPYRE approached single-molecule sensitivity for over 30 RNA fusion targets in a single reaction. The assay required only 1 ng RNA input, with a turn-around time of <4 hours from extracted RNA to result, opening the door to routine detection of RNA fusions from limited specimens. The simplicity and ease of use of ASPYRE, combined with its rapid turnaround time and analytical performance characteristics, provides a new tool for rapid and comprehensive treatment selection in NSCLC. Citation Format: Eleanor Gray, Justyna Mordaka, Efthimia Christoforou, Kristine von Bargen, Nicola Potts, Rebecca Palmer, Barnaby Balmforth. Low-cost, simple and rapid assay for single-molecule detection of gene fusions from RNA with ASPYRE technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4104.

CRISPR-Cas12a combination to alleviate the false-positive in loop-mediated isothermal amplification-based diagnosis of Neisseria meningitidis

BackgroundLoop isothermal amplification (LAMP) has recently been proposed as a point-of-care diagnostic tool to detect acute infectious pathogens; however, this technique embeds risk of generating false-positive results. Whereas, with abilities to accurately recognize specific sequence, the CRISPR/Cas12a can forms complexes with cognate RNA sensors and cleave pathogen’s DNA targets complimerntary to its cognate RNA, afterward acquiring the collateral activity to unbiasedly cut nearby off-target fragments. Therefore, if relevant fluorescent-quencher-nucleic probes are present in the reaction, the non-specific cleavage of probes releases fluorescences and establish diagnostic read-outs.MethodsThe MetA gene of N. meningitidis was selected as target to optimize the LAMP reaction, whereas pseudo-dilution series of N. meningitidis gemonics DNA was used to establish the detection limit of LAMP/Cas12a combination assay. The diagnostic performance of established LAMP/Cas12a combination assay was validated in comparation with standard real-time PCR on 51 CSF samples (14 N. meningitidis confirmed patients and 37 control subjects).ResultsIn relevant biochemical conditions, CRISPR-Cas12a and LAMP can work synchronously to accurately identify genetics materials of Nesseria menitigistis at the level 40 copies/reaction less than 2 h.ConclusionsIn properly optimized conditions, the CRISPR-Cas12a system helps to alleviate false positive result hence enhancing the specificity of the LAMP assays.

Detection of sex in adults and larvae of Leptinotarsa decemlineata on principle of copy number variation

The identification of sex in larvae of insects is usually challenging or even impossible, while in adults the sexual dimorphism is usually evident. Here, we used copy number analysis to develop a method of sex detection in Colorado potato beetle (Leptinotarsa decemlineata), which has an X0 sex determination system. The X linked gene LdVssc and autosomal gene LdUBE3B were identified as appropriate target and reference loci, respectively. The copy numbers (CNV) of LdVssc in males and females were estimated using standard droplet digital PCR (ddPCR) and real-time PCR (qPCR). With both methods, CNVs were bimodally distributed (BAddPCR = 0.709 and BAqPCR = 0.683) with 100% ability to distinguish females from males. The use of qPCR-based sex detection in a broad collection of 448 random CPB adults showed a perfect association (Phi = 1.0, p < 0.05) with the true sexes of adults, with mean CNV in females of 2.032 (SD = 0.227) and 0.989 in males (SD = 0.147). In the collection of 50 random 4th instar larvae, 27 females and 23 males were identified, consistent with the expected 1:1 sex ratio (p = 0.689). The method is suitable for sexing in all stages of ontogenesis. The optimal cost-effective application of the method in large populations requires the DNA extraction using CTAB, the qPCR assay in one biological replicate and three technical replicates of each marker, and the use of one randomly chosen male per run to calibrate calculation of CNV.