Ultrafast PCR Research Articles

Development of an ultrafast PCR to detect clinically relevant acquired vancomycin-resistance genes from cultured enterococci.

VRE are increasingly described worldwide. Screening of hospitalized patients at risk for VRE carriage is mandatory to control their dissemination. Here, we have developed the Bfast [VRE Panel] PCR kit, a rapid and reliable quantitative PCR assay for detection of vanA, vanB, vanD and vanM genes, from solid and liquid cultures adaptable to classical and ultrafast real-time PCR platforms. Validation was carried out on 133 well characterized bacterial strains, including 108 enterococci of which 64 were VRE. Analytical performances were determined on the CFX96 Touch (Bio-Rad) and Chronos Dx (BforCure), an ultrafast qPCR machine. Widely used culture plates and broths for enterococci selection/growth were tested. All targeted van alleles (A, B, D and M) were correctly detected without cross-reactivity with other van genes (C, E, G, L and N) and no interference with the different routinely used culture media. A specificity and sensitivity of 100% and 99.7%, respectively, were determined, with limits of detection ranging from 21 to 238 cfu/reaction depending on the targets. The Bfast [VRE Panel] PCR kit worked equally well on the CFX and Chronos Dx platforms, with differences in multiplexing capacities (five and four optical channels, respectively) and in turnaround time (45 and 16 minutes, respectively). The Bfast [VRE Panel] PCR kit is robust, easy to use, rapid and easily implementable in clinical microbiology laboratories for ultra-rapid confirmation of the four main acquired van genes. Its features, especially on Chronos Dx, seem to be unmatched compared to other tools for screening of VRE.

Assessing Different PCR Master Mixes for Ultrarapid DNA Amplification: Important Analytical Parameters.

The basic principles of ultrafast plasmonic PCR have been promulgated in the scientific and technological literature for over a decade. Yet, its everyday diagnostic utility remains unvalidated in pre-clinical and clinical settings. Although the impressive speed of plasmonic PCR reaction is well-documented, implementing this process into a device form compatible with routine diagnostic tasks has been challenging. Here, we show that combining careful system engineering and process control with innovative and specific PCR biochemistry makes it possible to routinely achieve a sensitive and robust "10 min" PCR assay in a compact and lightweight system. The critical analytical parameters of PCR reactions are discussed in the current instrument setting.

Handheld Ultra-Fast Duplex Polymerase Chain Reaction Assays and Lateral Flow Detection and Identification of Leishmania Parasites for Cutaneous Leishmaniases Diagnosis.

Early and accurate detection of infectious diseases is a key step for surveillance, epidemiology and control, notably timely disease diagnosis, patient management and follow-up. In this study, we aimed to develop handheld ultra-fast duplex PCR assays coupled to amplicon detection by lateral flow (LF) immunoassay to deliver a rapid and simple molecular diagnostic test for concomitant detection and identification of the main Leishmania parasites encountered in Tunisia. We selected two DNA targets to amplify L. major/L. tropica and L. infantum/L. tropica groups of species DNAs, respectively. We optimized the experimental conditions of a duplex ultra-fast PCR. The amplification is performed using a portable Palm convection PCR machine within 18 min, and the products are detected using an LF cassette within 10 min. The test allows the identification of the infecting species according to the position and number of test lines revealed. Tested on a selection of DNAs of representative Leishmania strains of the three studied species (N = 37), the ultra-fast duplex PCR-LF showed consistent, stable and reproducible results. The analytical limit of detection of the test was 0.4 pg for L. major, 4 pg for L. infantum and 40 pg for L. tropica.

Detection of SARS-CoV-2 using a laboratory-developed ultra-fast NextGenPCR test versus a conventional RT-PCR test

The reverse transcription polymerase chain reaction (RT-PCR) is considered the gold standard method for the detection of viruses in a clinic. The aim of this study was to compare the ability of conventional RT-PCR test (FTDTM SARS-CoV-2 Test) and laboratory-developed ultra-fast PCR test (NextGenPCRTM SARS-CoV-2 RT-PCR Reagent Kit) to detect the coronavirus SARS-CoV-2 causing COVID-19. A total of 318 nasopharyngeal swab specimens were collected from people under investigation for COVID-19. Despite the collection of two swab specimens from each patient and their different processing, the analysis showed an overall agreement of 95.9% between the conventional and laboratory-developed tests. The positive percentage agreement was 90.5% (114/126) and the negative percentage agreement was 99.5% (191/192). The ultra-fast NextGenPCR method does not require the isolation of RNA, provides a result of 20–96 specimens within 57–82 min after sampling, and offers a simple procedure of sample processing, analysis, and evaluation. Our results indicate that this method can be considered a potential diagnostic method for the detection of SARS-CoV-2 virus in hospitals, healthcare facilities, and research laboratories.

UtPCR: A Strategy for the Highly Specific and Absolutely Quantitative Detection of Single Molecules within Only Minutes.

Bloodstream infection is a major health problem worldwide, with extremely high mortality. Detecting infection in the early stage is challenging due to the extremely low concentration of bacteria in the blood. Digital PCR provides unparalleled sensitivity and can achieve absolute quantification, but it is time-consuming. Moreover, the presence of unavoidable background signals in negative controls poses a significant challenge for single-molecule detection. Here, we propose a novel strategy called "Ultrafast flexible thin tube-based droplet digital PCR (utPCR)" that can shorten the digital PCR process from 2 h to only 5 min, with primer annealing/extension time reduced from minutes to only 5 s. Importantly, the ultrafast PCR eliminates nonspecific amplification and thus enables single-molecule detection. The utPCR enabled the sensitive detection and digital quantification of E. coli O157 in the high background of a 106-fold excess of E. coli K12 cells. Moreover, this method also displayed the potential to detect rare pathogens in blood samples, and the limit of detection (LOD) could be as low as 10 CFU per mL of blood without false positive results. Considered ultrafast (<5 min) and highly sensitive (single-molecule detection), the utPCR holds excellent prospects in the next generation of molecular diagnosis.

A-376 Development of a 10-Minute Sample to Answer RT-PCR Respiratory Panel Performed on Point of Care (POC)

Abstract Background The COVID-19 pandemic has shown the importance of fast and sensitive PCR testing to identify SARS-CoV-2 (SC2) infected patients. Identifying infected individuals with a fast sample to answer test provides the opportunity to limit the spread of the virus and prescribe antimicrobial treatment in the same visit. Additionally, the post pandemic world has seen simultaneous outbreaks of RSV, influenza, and SC2, the so-called tridemic. These higher risk respiratory viruses can appear against a background of lower risk viruses such as rhinovirus, adenovirus, enterovirus, other corona viruses, and bacterial infections that present with similar symptoms and make triage challenging. Fast turnaround molecular testing along with multiplex detection of all key pathogens associated with flu-like symptoms is important for timely selection of treatment, counseling to contain the spread of disease, and provide public health officials information on prevalence of outbreaks. AMDI is developing a cloud-based, ultrafast, fully automated POC molecular technology that enables detection of up to 32 microbial targets in less than 10 min from sample to answer. A 15-second extraction-free hyperbaric heating (HBH) step is performed on the input swab sample to release nucleic acid and neutralize PCR inhibitors in the sample in seconds. The sample is directly transferred without dilution to a chamber where it mixes with dry RT-PCR reagents and then is dispatched to eight thin (&amp;lt;250 µm thick) PCR reaction cuvettes where rapid heat transfer can occur. This is followed by thermocycling on an instrument designed to achieve 10 sec per cycle and detect four fluorescent signals per cuvette. This study first evaluates the HBH method against nucleic acid purification assays using SC2 positive clinical samples. Next, a preliminary LoD on the AMDI system (HBH and fast thermocycling) was performed using inactivated SC2 and RSV whole organism in a nasal swab matrix. Methods To evaluate HBH as a sample prep method, 12 known SC2 positive remnant clinical samples in VTM or saline were diluted 1:1 with AMDI collection buffer, treated by HBH, tested by RT-PCR on QuantStudioTM and compared with the Roche LIAT. A preliminary SC2 and RSV LoD on the full AMDI system was determined by spiking dilutions of inactivated viruses into pooled nasal swab matrix, followed by treatment with HBH, and testing by RT-PCR using the ultra-fast thermocycler. Results In clinical sample testing HBH shows equal or greater sensitivity than LIAT, even for high Ct samples. HBH followed by RT-PCR detected 12/12 (100% sensitivity) samples while Roche LIAT detected 11/12 (92% sensitivity). When HBH is coupled with AMDI ultra-fast PCR thermocycling technology, an SC2 preliminary LoD of 500 copies/mL and an RSV preliminary LoD of 250 copies/mL was obtained in &amp;lt;9 min including a 60 second RT step. Conclusion HBH is an efficient and effective sample prep method and when coupled with ultrafast thermocycling provides sensitive PCR results in 10 minutes sample to answer.

Ultrafast Plasmonic Nucleic Acid Amplification and Real-Time Quantification for Decentralized Molecular Diagnostics.

Point-of-care real-time reverse-transcription polymerase chain reaction (RT-PCR) facilitates the widespread use of rapid, accurate, and cost-effective near-patient testing that is available to the public. Here, we report ultrafast plasmonic nucleic acid amplification and real-time quantification for decentralized molecular diagnostics. The plasmonic real-time RT-PCR system features an ultrafast plasmonic thermocycler (PTC), a disposable plastic-on-metal (PoM) cartridge, and an ultrathin microlens array fluorescence (MAF) microscope. The PTC provides ultrafast photothermal cycling under white-light-emitting diode illumination and precise temperature monitoring with an integrated resistance temperature detector. The PoM thin film cartridge allows rapid heat transfer as well as complete light blocking from the photothermal excitation source, resulting in real-time and highly efficient PCR quantification. Besides, the MAF microscope exhibits close-up and high-contrast fluorescence microscopic imaging. All of the systems were fully packaged in a palm size for point-of-care testing. The real-time RT-PCR system demonstrates the rapid diagnosis of coronavirus disease-19 RNA virus within 10 min and yields 95.6% of amplification efficiency, 96.6% of classification accuracy for preoperational test, and 91% of total percent agreement for clinical diagnostic test. The ultrafast and compact PCR system can decentralize point-of-care molecular diagnostic testing in primary care and developing countries.

Detection method for genetically modified potato using an ultra-fast PCR system.

Genetically modified (GM) potatoes having resistance to insects and viral diseases, low reducing sugar contents, and black spots for high quality continue to be developed. However, no GM potato has been approved as food or feed in the Republic of Korea as the country adheres to a zero-tolerance policy to unauthorized genetically modified organisms (GMOs). When the self-sufficiency rate is low, a detection method to assess GMOs in crops or other products is necessary. Therefore, a rapid method for two GM potato events (SPS-Y9 and EH92-527-1) using an ultra-fast PCR (UF-PCR) system has been developed, and its specificity, sensitivity, and applicability were demonstrated. UF-PCR can decrease the runtime of PCR by more than half of that needed in conventional methods. However, UF-PCR is not a common method for GMO analysis. This rapid detection method may be useful for GMO analyses in field conditions.

Sophisticated and precise: design and implementation of a real-time optical detection system for ultra-fast PCR.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) has become indispensable in the realm of disease nucleic acid screening and diagnostics, owing to its remarkable precision and sensitivity, in which the real-time fluorescence detection system plays an extremely critical role. To solve the problems of long time and slow speed of traditional nucleic acid detection, PCR systems are evolving towards ultra-rapid configurations. Nonetheless, most extant ultra-rapid PCR systems either depend on endpoint detection for qualitative assessments due to inherent structural or heating constraints or circumvent the challenge of adapting optical systems to expeditious amplification systems, resulting in potential shortcomings in assay efficacy, volume, or expense. Consequently, this study proposed a design of a real-time fluorescence detection system for ultra-fast PCR, capable of executing six channels of real-time fluorescence detection. Through the meticulous calculation of the optical pathway within the optical detection module, effective regulation of system dimensions and the cost was accomplished. By devising an optical adaptation module, the signal-to-noise ratio was enhanced by approximately 307% without compromising the PCR temperature alteration rate. Ultimately, by employing a fluorescence model that accounted for the spatial attenuation effect of excitation light, as proposed herein, fluorescent dyes were arranged to evaluate the repeatability, channel interference, gradient linearity, and limit of detection of the system, which proved that the system had good optical detection performance. Finally, the real-time fluorescence detection of human cytomegalovirus (CMV) under 9 min ultra-fast amplification was achieved by a complete ultra-fast amplification experiment, which further validated the potential of the system to be applied to rapid clinical nucleic acid detection.

Flap Endonuclease-Induced Steric Hindrance Change Enables the Construction of Multiplex and Versatile Lateral Flow Strips for DNA Detection.

A lateral flow strip (LFS) is an ideal tool for point-of-care testing (POCT), but traditional LFSs cannot be used for multiplex detection. Herein, a multiplex and versatile LFS based on flap endonuclease 1 (FEN1)-induced steric hindrance change (FISH-LFS) is proposed. In this method, multiplex PCR coupled with cascade invasive reactions was employed to yield single-stranded flaps, which were target-specific but independent of target sequences. Then, the amplicons were applied for FISH-LFS, and the single-stranded flaps would be efficiently captured by the complementary LFS-probes at different test lines. As flaps were cleaved from the specially designed hairpin probes, competition among flaps and hairpin probes would occur in capturing the probes at test lines. We enabled the hairpin probes to flow through the test lines while the flaps to stay at the test lines by making use of the difference in steric hindrance between hairpin probes and flaps. The assay is able to detect as low as two copies of blood pathogens (HBV, HCV, and HIV), to pick up as low as 0.1% mutants from wild-type gDNA, and to genotype 200 copies of SARS-CoV-2 variants α and β within 75 min at a conventional PCR engine. As the method is free of dye, a portable PCR engine could be used for a cost-effective multiplex detection on site. Results using an ultrafast mobile PCR system for FISH-LFS showed that as fast as 30 min was achieved for detecting three pathogens (HBV, HCV, and HIV) in blood, very suitable for POCT of pathogen screening. The method is convenient in operation, simple in instrumentation, specific in genotyping, and very easy in setting up multiplex POCT assays.

Advanced Technologies in On-Site Detection of Genetically Modified Products

Transgenic technology is significantly impacting life today. However, with the advancement of genetically modified technologies and the success of genetically modified product commercialization, new challenges have arisen for associated detecting technologies. The need for fast, precise, and portable systems for the on-site detection of genetically modified products has increased dramatically in recent years. This perspective examined the currently available technological support for portable immune biosensing, discussed a portable detection device for ultrafast PCR, and an on-site detection biosensor based on functional nucleic acid and superior detection devices in the field. Moreover, the on-site sequencing of genetically modified organisms was mentioned briefly. Lastly, the future outlook of genetically modified products detection was concluded and discussed in order to provide a comprehensive reference for future research and development in related fields.

Development of a rapid detection method for genetically modified rice using the ultra-fast PCR system.

Genetically modified (GM) rice varieties containing traits such as tolerance to abiotic stress and resistance against pests and diseases continue to be developed. However, contamination incidents from unauthorized GM rice varieties have been encountered. To date, no GM rice crop has been authorized for consumption and/or commercialization in Korea. Therefore, to enhance safety management of unauthorized genetically modified organisms (GMOs), accurate and reliable detection methods are needed to identify GMOs in crops or products. In this study, we developed rapid detection methods for GM rice events (Bt63, KMD1, Kefeng6, Kefeng8, and LLRice62) using ultra-fast PCR system. Ultra-fast PCR is a state-of-the-art technology and decreases PCR run-times dramatically. However, the ultra-fast PCR is not widely used in GMO analysis. Thus, we designed a detection method for five events of GM rice and confirmed them by performing specificity, sensitivity, and applicability assays. All results demonstrate that the ultra-fast PCR system is a specific, sensitive, and reliable method to identify and monitor GM rice events. Additionally, it can be utilized as a rapid and simple method for GMO analysis in crops or processed products. This study can be used as a reference for future research on new analysis methods of unauthorized GMOs.

Establishment and evaluation of a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument.

BackgroundThe coronavirus disease 2019 (COVID-19) pandemic is still raging worldwide. Efficient, fast and low-cost severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid detection methods are urgently needed.MethodsA rapid PCR temperature change mode was explored by moving the reaction tube between the independent temperature modules with large temperature differences and a portable ultra-fast real-time PCR instrument were developed. We established a rapid SARS-CoV-2 test method using the ultra-fast real-time PCR instrument, a China Food and Drug Administration-certified SARS-CoV-2 reagent and optimized reaction condition. The analytical and clinical performances of the rapid tests were evaluated by comparing with the standard SARS-CoV-2 tests.ResultsThe new temperature change mode can effectively shorten the amplification reaction time and be successfully used in the development of the ultra-fast real-time PCR instrument. The rapid SARS-CoV-2 test method was established and the time to yield results were greatly shortened from 81 min of the standard test to 31 min. Specificity of the rapid test was assessed and no non-specific amplification (0/63) was observed. The limits of detection of the rapid and standard tests were similar. Clinical performance was evaluated using 184 respiratory specimens from patients with suspected SARS-CoV-2 infection. The positive agreement between the rapid and standard tests was 100% (67/67), the negative agreement was 97.4% (114/117), and the kappa statistic was 0.965 (P<0.001). No significant differences in the Ct values for each target gene were observed between the rapid test and the standard test (P>0.05).ConclusionsWe had developed a 30-minute detection method for SARS-CoV-2 nucleic acid using a novel ultra-fast real-time PCR instrument. The rapid test method may impact on patient management.

Development of ultrafast PCR for rapid detection of buckwheat allergen DNA (fag e 1) in processed foods

Buckwheat allergy cases are increasingly reported worldwide, coinciding with the increasing consumption of buckwheat due to its high nutritional content. To detect the presence of buckwheat contaminants in food, we designed a buckwheat allergen-specific primer targeting Fag e 1 for use in an ultrafast PCR assay. This method amplified only common and Tartary buckwheat, and showed no cross-reactivity against 22 other plant samples. The limit of detection is 10 copies of plasmid DNA and 0.001% of buckwheat in wheat noodles, similar to that obtained from real-time PCR. In addition, this ultrafast PCR assay successfully detected buckwheat in processed foods within 25 min. The practical application of this method is similar to that of real-time PCR or ELISA. Thus, the ultrafast PCR assay we developed could be utilized as a highly specific and sensitive method for buckwheat detection in processed foods. • Ultrafast PCR for was developed to detect buckwheat allergen Fag e 1. • Buckwheat-specific primer sets amplified both common and Tartary buckwheat. • The limit of detection of this assay was 0.001% buckwheat in wheat. • Practical application in processed foods is similar to that of real-time PCR or ELISA. • This assay takes only 25 min to identify buckwheat in processed products.

Ultra-fast PCR method for the distinguishing between Miichthys miiuy and Sciaenops ocellatus.

The online version contains supplementary material available at 10.1007/s10068-021-00954-4.

Development of a Rapid On-Site Method for the Detection of Chicken Meat in Processed Ground Meat Products by Using a Direct Ultrafast PCR System

In this study, we developed a rapid on-site detection method by using direct ultrafast PCR coupled with a microfluidic chip to identify the presence of chicken meat in processed ground meat products. Chicken-specific PCR primer targeting mitochondrial 16S rRNA gene was newly designed, and its specificity was confirmed against 17 other animal species and 4 different chicken meat samples from different countries of origin. The sensitivity of the chicken-specific ultrafast PCR was 0.1 pg of chicken DNA. To evaluate the limit of detection of the direct ultrafast PCR method, different percentages of chicken meat mixed with pork or beef were prepared. The limit of detection of the direct ultrafast PCR method for the chicken meat-pork and chicken meat-beef mixtures was 0.1% for both raw meat and autoclaved meat. This method was used for 15 commercialized processed ground meat products. In this method, the target sequence was successfully amplified, and the presence of chicken meat in processed ground meat products was identified within approximately 25 min, including the time for sample preparation. Thus, our study shows that this developed direct ultrafast PCR method is a rapid and accurate method for on-site detection of chicken DNA in commercial food products.

Ultra Fast Real-Time PCR for Detection of Babesia gibsoni as Point of Care Test

Ultra Fast Real-Time PCR for Detection of Babesia gibsoni as Point of Care Test

Plasmon-Driven Ultrafast Photonic PCR

PCR is the gold standard for nucleic acid detection, which generally consists of three reaction stages (denaturation, annealing, and extension) and requires three corresponding temperatures. Although the commercialized thermocyclers use a Peltier block to heat the PCR solution in plastic tubes, they often lead to a heterogeneous temperature field and prolonged reaction time (∼hours). To address these issues, a plasmon-driven ultrafast photonic PCR technology has been proposed, which utilizes the photothermal effect of metallic nanomaterials (MNs) to heat the PCR solution under irradiation of incident light; here, the MNs play the role of energy converter from light energy to thermal energy.

Fast identification of Escherichia coli in urinary tract infections using a virulence gene based PCR approach in a novel thermal cycler

Uropathogenic Escherichia coli (UPEC) is the most common causal agent of urinary tract infections (UTIs) in humans. Currently, clinical detection methods take hours (dipsticks) to days (culturing methods), limiting rapid intervention. As an alternative, the use of molecular methods could improve speed and accuracy, but their applicability is complicated by high genomic variability within UPEC strains. Here, we describe a novel PCR-based method for the identification of E. coli in urine. Based on in silico screening of UPEC genomes, we selected three UPEC-specific genes predicted to be involved in pathogenesis (c3509, c3686 (yrbH) and chuA), and one E. coli-specific marker gene (uidA). We validated the method on 128 clinical (UTI) strains. Despite differential occurrences of these genes in uropathogenic E. coli, the method, when using multi-gene combinations, specifically detected the target organism across all samples. The lower detection limit, assessed with model UPEC strains, was approximately 104 CFU/ml. Additionally, the use of this method in a novel ultrafast PCR thermal cycler (Nextgen PCR) allowed a detection time from urine sampling to identification of only 52 min. This is the first study that uses such defined sets of marker genes for the detection of E. coli in UTIs. In addition, we are the first to demonstrate the potential of the Nextgen thermal cycler. Our E. coli identification method has the potential to be a rapid, reliable and inexpensive alternative for traditional methods.

Development and validation of ultrafast PCR assays to detect six species of edible insects

With the increasing use of insects as a food source, specific and sensitive detection methods for edible insects are required. To detect six insects (silkworm, mealworm, two-spotted cricket, rice grasshopper, white-spotted flower chafer beetle larvae, and rhinoceros beetle larvae) that have been approved for food consumption in Korea, an ultrafast PCR system was developed based on a microfluidic chip. Insect species-specific primer sets for the identification of the six edible insects were selected targeting the mitochondrial cytochrome c oxidase subunit I (COI) gene, and estimated for their specificity against 10 species of insects, which amplified only target insect species. The sensitivity of each primer set was independently validated in triplicate and was as little as 1 pg of DNA extracted from all insects. A total of 16 commercially processed products were used to verify the availability of these assays and to monitor the presence of labeled insect species. The ultrafast PCR assays developed in this study take approximately 20 min, and additionally confirm target species through post-PCR melt curve analysis. Thus, six species of edible insects could be rapidly and accurately identified in commercial food products using specific and sensitive ultrafast PCR methods.