Primer Dimers Research Articles

Validation of a Novel Strategy for Fluorescence Quenching for a Self-Quenching Fluorogenic Probe and Its Application for Visual Loop-Mediated Isothermal Amplification Detection During Food Safety Analysis

The self-quenching fluorogenic probe facilitates precise identification of LAMP (loop-mediated isothermal amplification) amplicons, unaffected by non-specific products resulting from primer dimers. However, low quenching efficiency by surrounding nucleobases leads to high background signal, posing significant challenges for visual inspection with the naked eye. The present study aims to identify an oligonucleotide sequence that is complementary to the self-quenching fluorogenic probe, and to employ the fluorescence super-quenching mechanism of double-stranded DNA to establish a visualization system for the LAMP assay. The results indicated that the incorporation of a sequence fully complementary to the probe could significantly reduce the system’s background fluorescence (p < 0.05). When the melting temperature exceeds room temperature, truncating the complementary sequence from the 3′ end does not compromise the probe’s quenching efficiency. The LAMP visualization system, using a 10–13-base complementary sequence of the loop primer-based probe, could effectively minimize background fluorescence and yield straightforward visual results post-reaction. Applied to rainbow trout and Atlantic salmon detection, the system detected 1 pg DNA in a closed-tube format. In conclusion, a suitable complementary sequence can reduce the background fluorescence of the self-quenching fluorogenic probe. Employing this sequence alongside the self-quenching fluorogenic probe to develop a low-background fluorescence LAMP system demonstrates great potential for successful visual detection and holds considerable promotional merit.

Harnessing non-standard nucleic acids for highly sensitive icosaplex (20-plex) detection of microbial threats.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. We benchmarked this assay using a low-cost, portable sequencing platform (Oxford Nanopore) on wastewater, soil, and human stool samples. Using parallelized multi-target TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay, there was 74% agreement on positive calls and 97% agreement on negative calls. Additionally, we show how sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish sub-species. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

Dye‐based recombinase‐aided amplification assay with enhanced sensitivity and specificity

AbstractObjectiveFluorescent recombinase‐aided amplification (RAA) assays are increasingly being used in the detection of a variety of pathogens and have the advantages of rapidity and simplicity and similar sensitivity and specificity, compared with real‐time PCR (qPCR) assays, but they require a complex probe design. To eliminate the addition of fluorescent probes for RAA, an EvaGreen dye‐based recombinase‐aided amplification (EvaGreen‐RAA) assay using self‐avoiding molecular recognition system (SAMRS) primers was developed.MethodsThe SAMRS primers effectively avoided the production of primer dimers, thus improving the detection sensitivity, while EvaGreen dye was used to quantitatively measure the amplified products in real time. Using Staphylococcus aureus (SA) and Listeria monocytogenes (LM) as examples, EvaGreen‐RAA with SAMRS primers was developed. As a reference and comparison, a traditional fluorescence probe RAA method and a RAA with SAMRS primers (SAMRS‐RAA) for detecting SA and LM were also investigated. Serial dilutions of recombinant plasmids were used to evaluate the sensitivity of the assays. Unenriched and enriched simulated milk samples were used to evaluate the limits of detection (LOD) of these methods. Using high‐resolution melting (HRM) was used to explore the sensitivity of the dual EvaGreen‐RAA assay.ResultsThe sensitivity of the fluorescent RAA method for detecting SA and LM was 10 copies/μL using plasmids and the sensitivity of the SAMRS‐RAA and EvaGreen‐RAA for detecting SA and LM plasmids was 1 copies/μL. The LOD values of the EvaGreen‐RAA for SA and LM in unenriched simulated milk samples were 100 and 50 CFU/mL, respectively, and the LOD value for both SA and LM using enriched simulated milk samples was 10 CFU/mL. EvaGreen‐RAA had linear amplification in real time in the range of 1–105 copies/μL of the plasmids of SA and LM. The sensitivity of the dual EvaGreen‐RAA assay for SA and LM was estimated to be 102 CFU/mL.ConclusionA real‐time quantitative EvaGreen‐RAA method for detecting SA and LM was developed, which eliminates the need to design complex RAA probes. This dye‐based RAA with SARMS primers provides a new strategy for simplifying fluorescence probe RAA and allowing the detection of multiple pathogens, which has many potential applications.

Multiplex Asymmetric PCR by Combining the Amplification Refractory Mutation System with the Homo-Tag-Assisted Nondimer System.

Asymmetric PCR is widely used to produce single-stranded amplicons (ss-amplicons) for various downstream applications. However, conventional asymmetric PCR schemes are susceptible to events that affect primer availability, which can be exacerbated by multiplex amplification. In this study, a new multiplex asymmetric PCR approach that combines the amplification refractory mutation system (ARMS) with the homo-Tag-assisted nondimer system (HANDS) is described. ARMS-HANDS (A-H) PCR utilizes equimolar-tailed forward and reverse primers and an excess Tag primer. The tailed primer pairs initiate exponential symmetric amplification, whereas the Tag primer drives linear asymmetric amplification along fully matched strands but not one-nucleotide mismatched strands, thereby generating excess ss-amplicons. The production of ss-amplicons is validated using agarose gel electrophoresis, sequencing, and melting curve analysis. Primer dimer alleviation is confirmed by both the reduced Loss function value and a 20-fold higher sensitivity in an 11-plex A-H PCR assay than in an 11-plex conventional asymmetric PCR assay. Moreover, A-H PCR demonstrates unbiased amplification by its allele quantitative ability in correct identification of all 31 trisomy 21 samples among 342 clinical samples. A-H PCR is a new generation of multiplex asymmetric amplification approach with various applications, especially when sensitive and quantitative detection is required.

Enhancing Loop-Mediated Isothermal Amplification through Nonpowered Nanoelectric Preconcentration.

The global threat posed by the COVID-19 pandemic has catalyzed the development of point-of-care (POC) molecular diagnostics. While loop-mediated isothermal amplification (LAMP) stands out as a promising technique among FDA-approved methods, it is occasionally susceptible to a high risk of false positives due to nonspecific amplification of a primer dimer. In this work, we report an enhancing LAMP technique in terms of assay sensitivity and reliability through streamlined integration with a nonpowered nanoelectric preconcentration (NPP). The NPP, serving as a sample preparation tool, enriched the virus concentration in samples prior to the subsequent LAMP assay. This enrichment enabled not only to achieve more sensitive assay but also to shorten the assay time for all tested clinical samples by ∼10 min compared to the conventional LAMP. The shortened assay time suppresses the occurrence of nonspecific amplification by not providing the necessary incubation time, effectively suppressing misidentification by false positives. Utilizing this technique, we also developed a prototype of the POC NPP-LAMP kit. This kit offers a streamlined diagnostic process for nontrained individuals, from the sample enrichment, transfer of the enriched sample to LAMP assays, which facilitates on-site/on-demand diagnosis of SARS-CoV-2. This development holds the potential to contribute toward preventing not only the current outbreak but also future occurrences of pandemic viruses.

Efficient detection of Streptococcus pyogenes based on recombinase polymerase amplification and lateral flow strip.

This article aims to establish a rapid visual method for the detection of Streptococcus pyogenes (GAS) based on recombinase polymerase amplification (RPA) and lateral flow strip (LFS). Utilizing speB of GAS as a template, RPA primers were designed, and basic RPA reactions were performed. To reduce the formation of primer dimers, base mismatch was introduced into primers. The probe was designed according to the forward primer, and the RPA-LFS system was established. According to the color results of the reaction system, the optimum reaction temperature and time were determined. Thirteen common clinical standard strains and 14 clinical samples of GAS were used to detect the selectivity of this method. The detection limit of this method was detected by using tenfold gradient dilution of GAS genome as template. One hundred fifty-six clinical samples were collected and compared with qPCR method and culture method. Kappa index and clinical application evaluation of the RPA-LFS were carried out. The enhanced RPA-LFS method demonstrates the ability to complete the amplification process within 6min at 33°C. This method exhibits a high analytic sensitivity, with the lowest detection limit of 0.908ng, and does not exhibit cross-reaction with other pathogenic bacteria. The utilization of RPA and LFS allows for efficient and rapid testing of GAS, thereby serving as a valuable method for point-of-care testing.

Progress in polymerase chain reaction technology and its contribution to the tuberculosis diagnostic ecosystem in India: A mini review

The advent of Polymerase Chain Reaction (PCR) has revolutionized the human molecular diagnostic scenario. Its ability to exponentially generate desired stretch of DNA from chromosomal or complimentary DNA has laid the foundation for sensitive and specific detection of a pathogen or an aberrant stretch of DNA. Importantly, PCR technology has helped generate diagnostic immunity from phenotypic variation of pathogenic targets. The technological evolution of conventional end-point PCR to its real time version further added clinically important features such as enhanced sensitivity and quantification of target DNA molecules. Two important chemistries became prominent in the real-time PCR domain. The intercalating dye-based chemistry comes with convenience, economy and feature such as melt curve analysis but also carries the disadvantage of overestimation of signal due to unwanted signal from primer dimers. Dual labelled probe on the other hand provides unprecedented sensitivity and specificity. Isothermal method of genome nucleic acid amplification has also evolved as a parallel and convenient method of amplification-based detection of nucleic acid targets. Tuberculosis diagnostics has immensely benefitted from these technologies. However, unmet need exists in sputum handling methods and in its adaptation for molecular diagnostics in resource limited settings. Significant dependence for diagnostic needs on the western world is a matter of concern in India. The country will benefit from rapid and indigenous research and development of molecular diagnostic solutions by way of formulating kits for rapid extraction of DNA and RNA from various clinical sources and developing detection technologies that can compete international products in its category. A solution to address sputum transportation, storage and DNA extraction that has enhanced advantages compared to current methods will also be helpful in the efforts to make India free from tuberculosis.

Highly specific detection of Neisseria gonorrhoeae based on recombinase polymerase amplification-initiated strand displacement amplification

Neisseria gonorrhoeae is the only pathogen that causes gonorrhea, and can have serious consequences if left untreated. A simple and accurate detection method for N. gonorrhoeae is essential for the diagnosis of gonorrhea and the appropriate prescription of antibiotics. The application of isothermal recombinase polymerase amplification (RPA) to detect this pathogen is advantageous because of its rapid performance, high sensitivity, and minimal dependency on equipment. However, this simplicity is offset by the risk of false-positive signals from primer-dimers and primer-probe dimers. In this study, RPA-initiated strand displacement amplification (SDA) was established for the detection of N. gonorrhoeae, and eliminated false-positive signals from primer–dimers and primer-probe dimers. The developed biosensor allows for the reduced generation of nonspecific RPA amplification through the design of enzyme cleavage sites on primers, introduction of SDA, and detection of the final product using a molecular beacon (MB). Using this system, the DNA double strand is transformed into single-stranded DNA following SDA, thereby providing a more suitable binding substrate and improving the efficiency of MB detection. Amplification can be conducted below 37 °C, and the process can be completed within 90 min. The limit of detection was determined to be 0.81 copies/μL. This system is highly specific for N. gonorrhoeae and exhibits no cross-reactivity with other common urogenital pathogens. The results of this study are consistent with those of real-time PCR performed on clinical specimens of urogenital secretions. In summary, the biosensor is a simple and specific detection method for N. gonorrhoeae.

Innovative Highly Specific Nucleic Acid Isothermal Detection Assay Based on the Polymerization-Coupled Endonuclease Activity of Prokaryotic DNA Polymerase I.

In this study, we developed an innovative highly specific nucleic acid isothermal detection assay based on prokaryotic DNA polymerase I with exquisitely designed fluorescent probes, achieving high sensitivity and 100% specificity within 30 min. The fluorescent nucleic acid probe was designed and constructed based on the specific flap cleavage endonuclease activity of prokaryotic DNA polymerase I (including the Bst, Bsu, Bsm, and Klenow DNA polymerases). The flap endonuclease activity depends on the length of the flap DNA and polymerization activity, which greatly reduces the false-positive rate caused by primer dimerization. This robust assay was also validated by the detection of rotavirus with great specificity and sensitivity. It could be a great alternative to qPCR in the field of point-of-care detection of pathogens.

Boron nitride nanoplate-based improvement of the specificity and sensitivity in loop-mediated isothermal amplification for Vibrio parahaemolyticus detection

BackgroundNucleic acid testing based on DNA amplification is gradually entering people's modern life for clinical diagnosis, food safety monitoring and infectious disease prevention. Polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) are the most powerful techniques that have been the gold standard for quantitative nucleic acid analysis. However, the high nonspecific amplification rate caused by the formation of primer dimers, hairpin structures and mismatched hybridization severely restricts their real-world applications. It is highly desirable to explore a way for improving the specificity and sensitivity of PCR and LAMP assays. ResultsIn this work, we demonstrated that a nanomaterial boron nitride nanoplate (BNNP), due to its unique surface properties, can interact with the main components of the amplification reaction, such as single stranded primers and Bst DNA polymerase, and increase the thermal conductivity of the solution. As a result, the presence of BNNPs dramatically improved the specificity of PCR and LAMP. And BNNPs maintained the specificity even after five rounds of PCR. Moreover, the sensitivity of LAMP was also enhanced by BNNPs, and the detection limit of BNNP-based LAMP was two orders of magnitude lower than that of classical LAMP. Then the BNNP-based LAMP was applied to detect Vibrio parahaemolyticus in contaminated seafood samples with high specificity and a 10-fold increase in sensitivity. SignificanceThis is the first systematic demonstration of BNNPs as a promising additive to enhance the efficiency and fidelity of PCR and LAMP amplification reactions, thereby greatly expanding the application of nucleic acid detection in a wide range of laboratory and clinical settings.

Simplified detection of the species of origin of antler velvets using single-stranded tag hybridization chromatographic printed-array strip.

In this study, we developed a convenient and easy-to-use origin identification method for antler velvets based on a simple DNA extraction technique and single-stranded tag hybridization chromatographic printed-array strip (STH-PAS). The primer sets used to detect Cervus elaphus, Rangifer tarandus, and 12S rRNA did not engage in non-specific reactions such as primer dimer formation. In both the triplex and singleplex assays, the sensitivity was < 1ng DNA. Moreover, Cervus elaphus DNA could be detected in OTC crude drug products. Although the detection sensitivity resulting from the simplified extraction was slightly lower than that obtained with extraction by conventional methods, the amount of DNA was sufficient even from a small sample. The choice of a triplex or singleplex assay will depend on the purpose of the test. For example, if it is important to determine whether the antler velvet is derived from Cervus elaphus or Rangifer tarandus, a triplex assay is appropriate. If it is necessary to explore whether antler velvet from Cervus elaphus is included in an OTC crude drug product, a singleplex assay using the Cervus elaphus primer set is informative. If it is necessary to explore whether powdered antler velvet includes counterfeit products (from Rangifer tarandus), a singleplex assay employing the Rangifer tarandus primer is appropriate. The singleplex assay detects minor components even at a 1,000:1 ratio. Our study thus demonstrated the utility of a method combining simple DNA extraction with STH-PAS for efficient identification of the origin of antler velvets.

An improved recombinase polymerase amplification assay for the visual detection of Staphylococcus epidermidis with lateral flow strips

Staphylococcus epidermidis is an opportunistic pathogenic microorganism that is an important cause of cross-infection in hospitals. The development of rapid and effective detection techniques is important for its control. The application of traditional identification and PCR-based methods is limited by their requirements for both laboratory instrumentation and trained personnel. To overcome this issue, we developed a fast detection approach for S. epidermidis that was based on recombinase polymerase amplification (RPA) and lateral flow strips (LFS). First, five pairs of primers were designed for molecular diagnosis using the sesB gene as the target, and were screened for their amplification performance and the formation of primer dimers. Specific probes were then designed based on the best primer pairs screened, which were susceptible to primer-dependent artifacts and generated false-positive signals when used for LFS detection. This weakness of the LFS assay was overcome by modifying the sequences of the primers and probes. The efficacy of these measures was rigorously tested, and improved the RPA-LFS system. Standardized systems completed the amplification process within 25 min at a constant temperature of 37 °C, followed by visualization of the LFS within 3 min. The approach was very sensitive (with a detection limit of 8.91 CFU/μL), with very good interspecies specificity. In the analysis of clinical samples, the approach produced results consistent with PCR and 97.78% consistent with the culture-biochemical method, with a kappa index of 0.938. Our method was rapid, accurate, and less dependent on equipment and trained personnel than traditional methods, and provided information for the timely development of rational antimicrobial treatment plans. It has high potential utility in clinical settings, particularly in resource-constrained locations.

Massively multiplex blocker displacement amplification (BDA) for the detection of measurable residual disease in acute myeloid leukemia.

e19045 Background: Next-generation sequencing (NGS) technologies, used to accomplish high throughput characterization of DNA, have an inherent error rate of between 0.1-1%. However, many important DNA sequences possess variant allele frequencies (VAFs) below this rate, potentially precluding their detection and identification by NGS. The previously published blocker displacement amplification (BDA) research technology has enabled selective amplification of sequences with variants, facilitating detection by NGS of these sequences with low VAFs at low read depths. Methods: Here we present the massively multiplex BDA library preparation method for the NGS-based detection of low VAF sequences. By optimizing our previously published Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE) algorithm, we designed a 4 tube assay, comprising up to ~2400 amplicons in each tube, and ~8300 total amplicons. Tube 1 was designed with the highest likelihood of on-target amplification and the lowest likelihood of dimerization or non-specific amplification. While Tube 1 targeted ~1000 regions covering the most-frequently observed mutations in current public databases of patients with acute myeloid leukemia (AML) across more than 200 genes, Tubes 2-4 were designed for enrichment of the full coding sequence of 63 genes. To highlight the performances of SADDLE, coverage uniformity, primer dimerization, and non-specific amplification of tube 1 were compared to those of a naively designed tube. The panel was further tested with contrived and reference samples to demonstrate its analytical performance. Results: The SADDLE algorithm design decreased the first pass amplicon dropout rate more than 10-fold, from 30% to ~2%, and further improved the overall on-target rate compared to the naively designed panel. Through SADDLE, we were able to achieve an 80% median coverage of 63 genes. The resulting panel has a bulk limit of detection (LoD) of below 0.1% VAF. Conclusions: Massively multiplex BDA enabled the NGS characterization of DNA targets with VAFs suitable for detection of AML measurable residual disease (MRD). This technology represents a 100-fold increase in current plexity for the detection of low VAF alleles over our previously published multiplex BDA technology, demonstrating the ability to perform high throughput sequencing of rare mutations.

Closed-tube visual detection of Atlantic cod (Gadus morhua) using self-quenched primer coupled with a designed loop-mediated isothermal amplification vessel.

Species adulteration has been widely revealed around the world, and the possible reasons include declining stocks in most source areas of the world, poor transparency in the global supply chain, and difficulty in distinguishing the features of processed products. The present work selected Atlantic cod (Gadus morhua) as a case study, and developed a novel loop-mediated isothermal amplification (LAMP) assay for Atlantic cod authentication, where a self-quenched primer and a newly designed reaction vessel were used to realize the endpoint visual detection of the target-specific products. A novel LAMP primer set was designed for Atlantic cod, and the inner primer BIP was selected to label the self-quenched fluorogenic element. The fluorophore's dequenching only occurred along with LAMP elongation for the target species. No fluorescence could be observed with both single-stranded DNA and partially complementary dsDNA of the non-target species. With the novel reaction vessel, both amplification as well as detection were operated in an enclosed device, and visual differentiation of Atlantic cod, negative control, and false positive generated from primer dimers was achieved. The novel assay has proved its specificity and applicability, and could detect as little as 1 pg of Atlantic cod DNA. Moreover, Atlantic cod adulteration as low as 10% could be detected in haddock (Melanogrammus aeglefinus), and no cross-reactivity was observed. The established assay could be a useful tool to detect mislabeling incidents involving Atlantic cod considering the advantages of speed, simplicity and accuracy. © 2023 Society of Chemical Industry.

Facilitation of Dye-Based Quantitative Real-Time Polymerase Chain Reaction with Poly(ethylene glycol)-Engrafted Graphene Oxide.

Quantitative real-time polymerase chain reaction (qPCR) is an important and extensively utilized technique in medical and biotechnological applications. qPCR enables the real-time detection of nucleic acid during amplification, thus surpassing the necessity of post-amplification gel electrophoresis for amplicon detection. Despite being widely employed in molecular diagnostics, qPCR exhibits limitations attributed to nonspecific DNA amplification that compromises the efficiency and fidelity of qPCR. Herein, we demonstrate that poly(ethylene glycol)-engrafted nanosized graphene oxide (PEG-nGO) can significantly improve the efficiency and specificity of qPCR by adsorbing single-stranded DNA (ssDNA) without affecting the fluorescence of double-stranded DNA binding dye during DNA amplification. PEG-nGO adsorbs surplus ssDNA primers in the initial phase of PCR, having lower concentrations of DNA amplicons and thus minimizing the nonspecific annealing of ssDNA and false amplification due to primer dimerization and erroneous priming. As compared to conventional qPCR, the addition of PEG-nGO and the DNA binding dye, EvaGreen, in the qPCR setup (dubbed as PENGO-qPCR) significantly enhances the specificity and sensitivity of DNA amplification by preferential adsorption of ssDNA without inhibiting DNA polymerase activity. The PENGO-qPCR system for detection of influenza viral RNA exhibited a 67-fold higher sensitivity than the conventional qPCR setup. Thus, the performance of a qPCR can be greatly enhanced by adding PEG-nGO as a PCR enhancer as well as EvaGreen as a DNA binding dye to the qPCR mixture, which exhibits a significantly improved sensitivity of the qPCR.

Improved Quantitative Real-Time PCR Protocol for Detection and Quantification of Methanogenic Archaea in Stool Samples

Methanogenic archaea are an important component of the human and animal intestinal microbiota, and yet their presence is rarely reported in publications describing the subject. One of the methods of quantifying the prevalence of methanogens is quantitative real-time PCR (qPCR) of the methanogen-specific mcrA gene, and one of the possible reasons for detection failure is usually a methodology bias. Here, we refined the existing protocol by changing one of the primers and improving the conditions of the qPCR reaction. As a result, at the expense of a slightly lower yet acceptable PCR efficiency, the new assay was characterized by increased specificity and sensitivity and a wider linear detection range of 7 orders of magnitude. The lowest copy number of mcrA quantified at a frequency of 100% was 21 copies per reaction. The other validation parameters tested, such as reproducibility and linearity, also gave satisfactory results. Overall, we were able to minimize the negative impacts of primer dimerization and other cross-reactions on qPCR and increase the number of not only detectable but also quantifiable stool samples-or in this case, chicken droppings.

Universal probe-based SNP genotyping with visual readout: a robust and versatile method.

Detection of single nucleotide polymorphisms (SNPs) is critical for personalized clinical diagnosis, treatment, and medication. Current clinical detection methods suffer from primer dimerization and require the redesigning of reaction systems for different targets, resulting in a time-consuming and laborious process. Here, we present a robust and versatile method for SNP typing by using tailed primers and universal small molecule probes in combination with a visualized lateral flow assay (LFA). This approach enables not only rapid typing of different targets, but also eliminates the interference of primer dimers and enhances the accuracy and reliability of the results. Our proposed universal assay has been successfully applied to the typing of four SNP loci of clinical samples to verify the accuracy and universality, and the results are consistent with those obtained by Sanger sequencing. Therefore, our study establishes a new universal "typing formula" using nucleic acid tags and small molecule probes that provides a powerful genotyping platform for genetic analysis and molecular diagnostics.

Development of a CRISPR/Cas9-integrated lateral flow strip for rapid and accurate detection of Salmonella

Rapid and accurate detection of foodborne pathogens is crucial for safeguarding human health. Herein, we constructed a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-integrated lateral flow strip (Cas9-LFS) for detecting Salmonella . A primer set targeting the fimA gene of Salmonella spp. was used to amplify all Salmonella serovars. After extraction, the genomic DNA of Salmonella was amplified with unlabelled forward primer and FITC-labelled reverse primer, thus producing numerous FITC-labelled amplicons that can be precisely recognized by Cas9 complexed with a specific single-guide RNA (sgRNA). When loaded on the LFS, the complexes of amplicons and Cas9/sgRNA can act as bonds to anchor gold nanoparticles to the test line, resulting in a visible color change. This design makes Cas9-LFS more powerful than conventional LFS in eliminating false-positives caused by primer dimers. By using Salmonella Typhimurium as a reference strain, Cas9-LFS achieved a limit of detection as low as 10 2 CFU/mL and showed good accuracy in distinguishing Salmonella -contaminated milk. We expect this unique strategy to open a new window for building versatile pathogen detection tools. • A CRISPR/Cas9-integrated LFS (Cas9-LFS) was constructed for accurate and visual detection of Salmonella. • Primer-dimer interference that is inevitable in conventional amplifications can be totally overcome with the method. • As low as 10 2 CFU/mL of Salmonella can be well measured with the constructed method. • Salmonella -contaminated milk samples have been measured with considerable accuracy.

Thermophilic helicase-dependent amplification-based CRISPR/Cas12a system: Detection of stx2 in Escherichia coli O157:H7 by controlling primer dimers

Backgrounds: To overcome the limitation of polymerase chain reaction (PCR), isothermal amplification methods such as thermophilic helicase-dependent amplification (tHDA) have been developed. However, formation of primer dimer due to the single amplification temperature are major problems of tHDA. When cross-dimerization of forward and reverse primer occurred, false-positive results can be found on the lateral flow assay (LFA) which is one of the major detection methods widely used as a point of care diagnosis. Therefore, specific method of detecting only the target amplicon is required. ResultsIn this study, a tHDA-based CRISPR/Cas12a system was developed to detect low levels of Escherichia coli O157:H7 in fresh salad mix without the false-positive results produced by primer dimers. For the comparison of the effect in eliminating false-positive results by CRISPR/Cas12a system, LFA was also evaluated. The tHDA-based CRISPR/Cas12a system detected as low as 101 CFU/mL E. coli O157:H7 in bacterial pure culture. In LFA false-positive results were produced due to the primer dimer, whereas the primer dimer produced by tHDA was not detected in the CRISPR/Cas12a system. These results indicated that the CRISPR/Cas12a system eliminated the formation of primer dimer. In fresh salad mix, the tHDA-based CRISPR/Cas12a system combined with the filter concentration method detected 103 CFU/g E. coli O157:H7. ConclusionThis study was the first to amplify stx2 of E. coli O157:H7 with tHDA as an isothermal amplification method and detected the amplicon without false-positive results by combining tHDA with CRISPR/Cas12a. Therefore, this study showed great potential for detecting low levels of E. coli O157:H7 present in fresh salad mix.

Controlled Rehydration of Dried Reagents for Robust Multiplex Digital PCR.

Digital polymerase chain reaction (dPCR) is emerging as a powerful method for nucleic acid detection due to its unprecedented sensitivity and precision. However, most current dPCR platforms are inherently limited by their low multiplexing ability due to primer-pair cross interactions and spectral overlap of available fluorophores. Here, we present a novel and robust method for multiplexing dPCR that is free from primer dimerization and fluorescence channel number limitation, enabling highly precise and multiplexed detection of nucleic acid targets. By prestoring target-specific primers and probes in different storage chambers, the method physically separates reactions and thus avoids the primer-pair cross interactions and spectral overlap of different fluorescent probes that usually occur within a single-tube reaction. Furthermore, a dissolvable delay valve (DDV) is embedded between each pair of the reagent prestorage chamber and reaction microwell array. Such a DDV configuration allows full reconstitution of the prestored reagents and then generates a uniform concentration distribution of the reconstituted reagents across the entire reaction microwell array, which is favorable for achieving reliable and robust multiplex dPCR assays. We demonstrated the feasibility of this method by performing an eight-plex dPCR assay targeting the seven most common point mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) and a reference sequence (wild-type KRAS allele).