Non-specific Amplification Research Articles

Highly Precise and Sensitive Polymerase Chain Reaction Using Mesoporous Silica-Immobilized Enzymes.

A highly precise and sensitive technology that enables DNA amplification/detection from minimal amounts of nucleic acid is expected to find applicability in genetic testing involving small amounts of samples. The use of a free enzyme in conventional DNA amplification techniques, such as the polymerase chain reaction (PCR), frequently causes side reactions (i.e., nonspecific DNA amplification) when ≤103 substrate DNA molecules are present, thereby preventing selective amplification of the target DNA. To address this issue, we have developed a novel DNA amplification system, mesoporous silica-enhanced PCR (MSE-PCR), which involves the immobilization of a thermostable DNA polymerase from Thermococcus kodakaraensis (KOD DNA polymerase) into highly ordered nanopores of the mesoporous silica to control the reaction environment around the enzyme. In the MSE-PCR system using immobilized KOD DNA polymerase, such nonspecific DNA amplification was remarkably inhibited under the same conditions. Furthermore, the optimization of mesoporous silica pore sizes enabled selective and efficient DNA amplification from DNA substrates at the single-molecule level, i.e., one ten-thousandth of the amount of substrate DNA required for a DNA amplification reaction using a free enzyme. The results obtained in this study have shown that the nanopores of mesoporous silica can inhibit nonspecific reactions in DNA amplification, thereby considerably improving the specificity and sensitivity of the DNA polymerase reaction.

Beyond Domestic Cats: Environmental Detection of Sporothrix brasiliensis DNA in a Hyperendemic Area of Sporotrichosis in Rio de Janeiro State, Brazil.

In Brazil, sporotrichosis has transitioned from a rural to urban disease, driven by a shift in the initiation of infection from the accidental inoculation of organic matter to the traumatic implantation of the fungus by cats. Since the emergence of zoonotic sporotrichosis caused by Sporothrix brasiliensis, investigations have largely ignored the environmental habitat of the pathogen due to its association with domestic cats. Therefore, we investigated 18 environmental samples collected from rural areas of two cities where zoonotic sporotrichosis is endemic, but where domestic cats are scarce. We utilized traditional culture methods, and samples were also examined with two molecular methods used for the clinical diagnosis of sporotrichosis: a nested-PCR targeting the ITS region and a species-specific PCR targeting the calmodulin gene. No Sporothrix colonies were identified by traditional culture methods. However, the nested-PCR and the species-specific PCR for S. brasiliensis were positive for 18 and 5 samples, respectively. Sequencing revealed that positive results with the nested-PCR were due to non-specific amplification of other Ophiostomatales DNA, rather than Sporothrix spp. Three of the five amplicons from the species-specific PCR were suitable for sequencing and confirmed the presence of S. brasiliensis DNA. Hence, we confirmed that S. brasiliensis, as with other Sporothrix species, has an environmental habitat. Our findings underscore the challenges of nested-PCR for Sporothrix environmental studies and highlight that sequencing must follow PCR protocols to definitively identify Sporothrix spp. in environmental samples.

Non-nucleic acid extraction and ultra-sensitive detection of African swine fever virus via CRISPR/Cas12a.

Early diagnosis of the African swine fever virus (ASFV) is the main preventive measure for ASFV. Here, we developed a fluorescent biosensor and lateral flow assay (LFA) strip based on direct PCR combined with CRISPR/Cas12a system for ASF. Direct PCR can simultaneously split samples and efficiently amplify without sacrificing sensitivity, which eliminated the steps of nucleic acid extraction. Furthermore, by the CRISPR/Cas12a, the biosensor addressed false positives caused by non-specific amplification and had high sensitivity with the actual limit of detection (LOD) of 7.6×10-4 ng·μL-1 (4 copies·μL-1). In addition, the strategy was built on the lateral flow assay (LFA) strip to achieve visual and portable detection for point-of-care testing. Moreover, the biosensor by a fluorometer and LFA strip showed a high accuracy to rival qPCR in actual sample detection. Therefore, the biosensor is an ultra-sensitive and specific tool that can replace traditional methods. KEY POINTS: • No nucleic acid extraction, direct PCR-simplified steps, and reduced time and cost • CRISPR/Cas12a solved the false positives caused by nonspecific amplification • The combination of the LFA strip and biosensor is more convenient for POC detection.

Responses of soil ammonia-oxidizing microorganisms to simulated nitrogen deposition in a natural Castanopsis carlesii forest

Subtropical region of China is one of the global hotspots receiving nitrogen deposition. Nitrogen deposition could affect the abundance and community structure of ammonia oxidizers including ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA) and complete ammonia oxidizer (comammox Nitrospira), with consequences on soil nutrient cycling that are driven by microorganisms. There is limited understanding for the newly discovered comammox Nitrospira in the subtropical forest soils. Here, we investigated the effect of simulated N deposition on abundances of soil ammonia oxidizers in the Castanopsis fargesii Nature Reserve in Xinkou Town, Sanming City, Fujian Province, China. Soil samples were collected from the field plots which received long-term nitrogen deposition with different dosages, including: CK, no additional treatment; LN, low nitrogen deposition treatment, dosage of 40 kg N·hm-2·a-1; and HN, high nitrogen deposition treatment, dosage of 80 kg N·hm-2·a-1. After 8-year treatment, simulated N deposition decreased soil pH and organic matter content, and increased nitrate content. We failed to amplify the amoA gene of AOB in the tested soils. High nitrogen deposition increased the abundance of AOA, but did not affect the abundance of comammox Nitrospira clade A and clade B. The ratio of comammox Nitrospira to AOA decreased with N addition, indicating that N addition weakened the role of comammox Nitrospira in nitrification in the subtropical forest soils. However, there were strong non-specific amplifications for both comammox Nitrospira clades A and B, highlighting the demand for the development of high coverage and specificity primers for comammox Nitrospira investigations in the future. The abundance of comammox Nitrospira clade A was positively correlated with total nitrogen (TN) and NH4+ concentration, while that of clade B was positively associated with soil organic carbon (SOC), TN and NH4+ Concentration. Overall, our findings demonstrated that simulated N deposition increased the relative importance of AOA in nitrification in the natural Castanopsis carlesii forest soil. These findings could provide theoretical support in coping with global change and N deposition in these regions.

Detection of a single nucleotide polymorphism for insecticide resistance using recombinase polymerase amplification and lateral flow dipstick detection

Modern methods of single nucleotide polymorphism (SNP) detection require high-throughput or automated technologies that preclude field deployment. To develop a low-resource SNP detection system, we evaluated Recombinase Polymerase Amplification coupled with lateral flow dipstick (RPA-LFD) detection, targeting, as a proof-of-concept, an SNP (F1534C) that leads to pyrethroid resistance in Aedes aegypti mosquitoes. Primers and probes were designed to contain variable numbers and positions of mismatches and tested for their ability to amplify synthetic gene fragments with either wild-type or the single mismatch mutant gene sequence. Our results demonstrated, for the first time, that detection of a single nucleotide change could be achieved in this low-resource format by performing tenfold serial dilutions of gene fragments, which reduced non-specific amplification at high template concentrations. Using a forward primer SNP detection approach, removing the reverse primer enhanced discrimination with single, double and three scattered mismatches, with sensitivity decreasing 10-fold per additional mutation; a single 3′ mismatch provided the greatest (3-log) difference in sensitivity between the two variants. Similar results were observed using a reverse probe-based mismatch approach, however, the forward primer variants showed greater impacts on RPA efficiency. These results demonstrate significant potential for SNP mutant detection using a rapid 15-minute low-resource test.

A high-throughput analytical method for multiple DNA targets based on microdroplet PCR coupled with DGGE

We developed a novel approach to analyze multiple DNA targets based on microdroplet PCR coupled with denaturing gradient gel electrophoresis (MPDG) to achieve high-throughput detection of biological samples. The target DNAs were preamplified using specific primers. Subsequently, the preamplified products were separated into individual microreactors for parallel amplification with high efficiency, avoiding the interference of different primers and templates, and preventing inconsistent amplification efficiency and non-specific amplification. The final products were analyzed using denaturing gradient gel electrophoresis (DGGE). Using genetically modified (GM) maize as samples, the MPDG method could be used for the simultaneous detection of three DNA targets with an absolute limit of detection of 0.5% (w/w), with no cross reaction with other non-GM samples. The simulated sample assay of MPDG suggested that the method is suitable for practical application. The MPDG approach, with high sensitivity and specificity, could play a crucial role in the field of nucleic acid detection.

Development and validation of a direct PCR based assay for the detection of Colletotrichum species on chili seeds

An investigation was executed to detect Colletotrichum truncatum (synonymous C. capsici) and C. coccodes in solo or as a disease complex through direct PCR (dPCR) in anthracnose-infected chili seeds. Direct PCR was performed with C. coccodes and C. truncatum-specific markers and Tris-EDTA buffer aliquots (obtained from infected seeds soaked up to five hours) as source of template DNA. This method efficiently and specifically detected the respective species in seeds with minimum 2.5% infection, yielding species-specific ∼500 bp (C. truncatum) and ∼340 bp (C. coccodes) fragments without any non-specific amplification with other mycoflora. Further, the seeds used in the experiment were tested for their germination efficiency along with a complete set of dried seeds as control. Among the soaked seeds, germination frequency ranged between 50 (infected seeds) to 100% (healthy seeds) without any significant loss in germination, confirming the sustainability of the current protocol. We recommend the use of direct PCR from soaked seeds without prior DNA extraction as a cost-effective and quick method for detecting the pathogen directly from infected seeds in fields.

A comparison of mRNA sequencing (RNA-Seq) library preparation methods for transcriptome analysis

BackgroundmRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis.ResultsWe observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated.ConclusionsTruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime with regards to the detected number of transcripts and splicing events among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.

Assessment of the Real-Time PCR Method Claiming to be Specific for Detection and Quantification of the First Commercialised Genome-Edited Plant

A real-time PCR method was recently published with a claim to be specific for the detection and identification of some genome-edited oilseed rape (OSR) lines commercialised in North America. The method was designed to detect a single base mutation in the AHAS1C gene, which confers herbicide tolerance. The authors claim that the method is event-specific for the genome-edited OSR line 5715 and fulfils all requirements for GMO analytical methods according to EU regulations. We have thoroughly assessed the method in relation to the minimum performance requirements (MPR) established by the European Network of GMO Laboratories (ENGL). The method was found to be sufficiently sensitive and robust when tested with pure genomic DNA of the OSR line 40 K. However, our results show that the method is not event-specific and detects also OSR lines carrying the same point mutation caused by somaclonal variation. Moreover, impaired robustness was observed using non-modified genomic DNA at the amount specified in the original protocol. Significant non-specific PCR amplifications with PCR products as non-target template DNA and with genomic DNA from numerous OSR varieties as well as from wild radish were found by three ISO/IEC 17025 accredited reference laboratories in tests using different master mixes and PCR cycler models. The assessment shows that the method does not meet the MPR for qualitative PCR methods and therefore is not fit-for-purpose for official controls of genetically modified products in the EU. Suggestions are provided for conditions under which analytical methods for genome-edited organisms should be validated.

Closed dumbbell mediated isothermal amplification of nucleic acids for DNA diagnostic assays.

Nucleic acid amplification tests have been widely applied in clinical diagnostics, food safety monitoring, and molecular biology. As a well-established isothermal amplification method, Loop-mediated isothermal amplification (LAMP) has gained recognition. However, the need for specifically designed four to six primers and non-specific amplification pose challenges for further application of LAMP based detection methods. Here, a novel isothermal amplification method, termed closed dumbbell mediated isothermal amplification (CDA) of nucleic acids, was developed. The primers are easily designed by adding two different parts of middle sequence to the canonical PCR primers at 5'-ends. CDA method was demonstrated in detecting MERS-CoV orf1a gene and H1N1 gene fragments with merits of short core primer, simple primer design process and high amplification efficiency. In addition, CDA showed excellent amplification efficacy over LAMP and competitive annealing mediated isothermal amplification (CAMP) by slight modification of primers targeting at same sequence. Furthermore, real-time and HNB based colorimetric CDA detection of Shigella were developed for practical application, both exhibited 100% success. In all, the developed CDA method with high specificity, simplicity, efficiency and rapidity has shown its great potential for point of care nucleic acids diagnostic.

Loop-mediated isothermal amplification-based nucleic acid lateral flow assay for the specific and multiplex detection of genetic markers

In this study, a loop-mediated isothermal amplification-based nucleic acid lateral flow assay (LAMP-NALFA) system was developed for the specific and multiplex detection of genetic markers at a low cost. In principle, the LAMP reaction was optimized to generate a single-stranded sequence in the LAMP product, which was designed to serve as a barcode sequence and to specifically bind to the DNA capture on a NALFA strip. As a target genetic marker, the Salmonella enterotoxin (stn) gene was chosen and determined down to 9 aM (∼5.44 copies/μL). Importantly, the proposed system clearly discriminated the specific target amplification products from non-specific amplification products resulting from primers or non-target nucleic acids, proving the high selectivity of the LAMP-NALFA system. Furthermore, the practical applicability of the system was demonstrated by detecting Salmonella bacteria in Luria-Bertani medium, drinking water, and eggshells, with a limit of detection of 1.6 CFU. Finally, two different bacteria (Salmonella and Staphylococcus) were simultaneously determined by the multiplex LAMP-NALFA system. It is expected that the LAMP-NALFA system could be used in a point-of-care setting for the detection of bacteria or viruses, consequently improving both individual and public health.

Visual detection of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) simultaneously by duplex loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is often confounded by the non-specific amplification arising from primer dimers, off-target priming, and other artifacts. Precipitation of the DNA produced during LAMP with the use of specific fluorescently labeled probe has proved the effectiveness in specific detection. Herein, two fluorophores (ROX and FAM) were attached to the primers S-LB-6 and R-FIP for Atlantic salmon and rainbow trout, respectively, which are self-quenched in unbound state and become de-quenched after binding to the dumbbell-shaped DNA specifically. The DNA precipitation and appearance of small sediment took 10 s of centrifugation at 1000 g, by adding polyethylenimine (PEI) 600. Each target species was specifically amplified with the predicted color of PEI-DNA sediment, namely red for Atlantic salmon, green for rainbow trout, and pale yellow for mixed species. The optimized duplex LAMP system has proved its specificity and can detect as little as 1 ng DNA in visual detection.

CRISPR-Cas14a-integrated strand displacement amplification for rapid and isothermal detection of cholangiocarcinoma associated circulating microRNAs

Circulating microRNAs (miRNA) can serve as key biomarkers for early diagnose of cholangiocarcinoma. Herein, an assay that uses circulating miRNA to trigger strand displacement amplification (SDA) and a CRISPR-Cas14a system to report the SDA process has been developed. In the proposed method, SDA directly amplifies miRNAs without reverse transcription. The reporter, CRISPR-Cas14a, can reduce the risks of non-specific amplification and offers a sequential amplification that improves the sensitivity for miRNA detection. The assay, termed Cas14SDA, can discriminate miRNAs with similar sequences and can detect as low as 680 fM miR-21 (miRNAs overexpressed in cholangiocarcinoma) within 1 h. In particular, Cas14a was efficiently activated by a single-stranded SDA amplicon which improved the sensitivity by 2.86 times compared to that using Cas12a. This research has demonstrated that the Cas14SDA assay can discriminate cholangiocarcinoma patients from healthy donors by testing miR-21 in their blood samples. The Cas14SDA assay developed broadens the toolbox for miRNA biomarker analysis.

An improved nucleic acid sequence-based amplification method mediated by T4 gene 32 protein.

The uptake of Nucleic Acid Sequence-Based Amplification (NASBA) for point of care testing may be hindered by a complexity in the workflow due the requirement of a thermal denaturation step to initiate the cyclic isothermal amplification before the addition of the amplification enzymes. Despite reports of successful enhancement of other DNA and RNA amplification methods using DNA and RNA binding proteins, this has not been reported for NASBA. Here, three single-stranded binding proteins, RecA, Extreme Thermostable Single-stranded binding protein (ET SSB) and T4 gene gp32 protein (gp32), were incorporated in NASBA protocol and used for single pot, one-step NASBA at 41 °C. Indeed, all SSBs showed significantly improved amplifications compared with the 2-step process, but only gp32 showed no non-specific aberrant amplification, and slightly improved the time-to-positivity in comparison with the conventional NASBA. For synthetic HIV-1 RNA, gp32 was found to improve the time-to-positivity (ttp) by average of 13.6% of one-step NASBA and 6.7% of conventional NASBA for the detection of HIV-1 RNA, showing its potential for simplifying the workflow as desirable for point of care applications of NASBA.

Development and validation of multiplex SYBR Green real-time PCR assays for detection and molecular surveillance of four tick-borne canine haemoparasites

Two multiplex SYBR Green based real-time PCR assays were standardized and evaluated to detect DNA from four canine haemoparasites (Babesia gibsoni, Babesia vogeli, Ehrlichia canis and Hepatozoon canis), along with internal controls from dogs from selected districts of Punjab state, India. Amplicons of 126 bp, 337 bp, 234 bp and 106 bp corresponding to B. gibsoni (18S rRNA gene), B. vogeli (18S rRNA gene), E. canis (virB9 gene), and H. canis (18S rRNA gene) were obtained, without any non-specific amplification. Microscopic evaluation of 200 blood samples from dogs revealed the prevalence of B. gibsoni, E. canis and H. canis as 1.5%, 1.5% and 1.0%, respectively, while with the multiplex real-time PCR assays the values for B. gibsoni, B. vogeli, E. canis, and H. canis were 8.0%, 1.5%, 3.5% and 23.5%, respectively, with concurrent infections of B. gibsoni and H. canis (3.5%); E. canis and H. canis (2.0%) and B. gibsoni, B. vogeli, E. canis, and H. canis (0.5%). The diagnostic sensitivity of the multiplex real-time PCR assays with respect to microscopy in the detection of B. gibsoni, E. canis and H. canis was 100% while the specificity for B. gibsoni, B. vogeli, E. canis, and H. canis was 93%, 100%, 98% and 77%, respectively, revealing the respective strength of agreement as ″fair″, ″slight″, ″moderate″ and ″slight″ by kappa value statistics, and the data were statistically significant, for detection of B. gibsoni and E. canis infections, by Fisher's exact test. The analytical sensitivity of the multiplex PCR assays in detection of DNAs was 8.59 × 105 and 9.9 × 106 copies for B. vogeli and E. canis, respectively, and 1.15 × 106 and 3.41 × 105 copies for B. gibsoni and H. canis, respectively. Assessment of risk factors viz. age, sex, breed, season and locations showed no significant association with the prevalence of these haemoparasites except for B. vogeli, E. canis and H. canis where significant associations were found for location, age and breed, respectively by multiplex real-time PCR assays.

Multiplex, Real-Time, Point-of-care RT-LAMP for SARS-CoV-2 Detection Using the HFman Probe.

Viral evolution impacts diagnostic test performance through the emergence of variants with sequences affecting the efficiency of primer binding. Such variants that evade detection by nucleic acid-based tests are subject to selective pressure, enabling them to spread more efficiently. Here, we report a variant-tolerant diagnostic test for SARS-CoV-2 using a loop-mediated isothermal nucleic acid-based amplification (LAMP) assay containing high-fidelity DNA polymerase and a high-fidelity DNA polymerase-medicated probe (HFman probe). In addition to demonstrating a high tolerance to variable SARS-CoV-2 viral sequences, the mechanism also overcomes frequently observed limitations of LAMP assays arising from non-specific amplification within multiplexed reactions performed in a single “pot”. Results showed excellent clinical performance (sensitivity 94.5%, specificity 100%, n = 190) when compared directly to a commercial gold standard reverse transcription quantitative polymerase chain reaction assay for the extracted RNA from nasopharyngeal samples and the capability of detecting a wide range of sequences containing at least alpha and delta variants. To further validate the test with no sample processing, directly from nasopharyngeal swabs, we also detected SARS-CoV-2 in positive clinical samples (n = 49), opening up the possibility for the assay’s use in decentralized testing.

Rapid, Visual, and Sequence-Specific Detection of Salmonella in Egg Liquid with vis-NEAA, a CRISPR/Cas12 Empowered New Strategy.

Salmonella is one of the main pathogenic factors that cause foodborne diseases. Rapid and accurate detection of Salmonella in food is of great importance to ensure food safety. Nicking enzyme-assisted amplification (NEAA) is one of the promising isothermal amplification methods finishing the in vitro amplification in ∼10 min; however, it suffers from nonspecific amplification a lot (∼70% products are noises). In this paper, we introduced CRISPR/Cas12a to specifically recognize the NEAA amplicons and transduce the signals into turned-on fluorescent visual readouts (vis-NEAA). Impressively, with this method, the high efficiency of NEAA has been taken great advantage and the nonspecific products were successfully bypassed at the same time. In comparison to NEAA-gel electrophoresis, vis-NEAA showed complete fidelity toward the presence of specific products, while for real-time PCR, it possesses equivalent sensitivity and specificity but saves ∼80% of the time. A level of 80 CFU/mL Salmonella in spiked eggs can be detected on-site in ∼20 min.

Establishment of a rapid method for skipjack tuna (Katsuwonus pelamis) authentication using molecular beacons in loop-mediated isothermal amplification

One major drawback to the traditional loop-mediated isothermal amplification (LAMP) detection methods is the increased likelihood of detecting false-positive signals derived from non-specific amplification. Molecular beacon (MB) is increasingly being used in many applications and the MB-LAMP assay has proved itself as a target-specific method. The present work selected skipjack tuna as a case study, and developed a novel MB-LAMP assay for rapid species authentication. Specifically, the optimal MB structure includes 13 nucleobases in the loop region (binding specifically to loop primer LF) and 5 nucleobases in the stem region. For the established MB-LAMP assay, in the presence of the amplicons, the MB probe LFP-1 hybridizes to its target and forms a double helix. The change in conformation separates the quencher from the fluorophore, thereby resulting in the fluorescence release. The novel MB-LAMP assay has proved its specificity and can detect as little as 0.5 pg of skipjack tuna DNA.

A New Real-Time PCR Assay for Detecting Fungi in Genus Ceratocystis.

The genus Ceratocystis contains several significant plant pathogens, causing wilt and canker disease on a wide range of plant species. There are >40 known species of Ceratocystis, some of which are becoming increasingly important in agricultural or natural ecosystems. The diagnostic procedures for most Ceratocystis species rely on time-consuming and labor-intensive culturing approaches. To provide more time-efficient and sensitive molecular diagnostic tools for Ceratocystis, a generic TaqMan real-time PCR assay was developed using the ITS gene. This novel two-probe TaqMan assay amplified DNA from all tested Ceratocystis species. Some nonspecific amplification of a few species from closely related genera was observed under certain conditions; however, these false-positive detections could be ruled out using the additional PCR primers developed for further sequence-based identification of the detected species. The assay was found to be highly sensitive, as it detected 0.2 pg/μl of Ceratocystis DNA in water as well as in host DNA matrix. Further validation with artificially inoculated fig stem tissue demonstrated that the assay was also able to effectively detect the pathogen in infected asymptomatic stem tissue. This newly developed real-time PCR assay has practical applications in biosecurity, conservation, and agriculture; it will enable the detection of Ceratocystis species directly from plant material to facilitate more sensitive screening of imported plant germplasm, and allow rapid tracking of pathogens in the case of disease outbreaks.

An encodable multiplex microsphere-phase amplification sensing platform detects SARS-CoV-2 mutations

SARS-CoV-2 variants of concern (VOCs) contain several single-nucleotide variants (SNVs) at key sites in the receptor-binding region (RBD) that enhance infectivity and transmission, as well as cause immune escape, resulting in an aggravation of the coronavirus disease 2019 (COVID-19) pandemic. Emerging VOCs have sparked the need for a diagnostic method capable of simultaneously monitoring these SNVs. To date, no highly sensitive, efficient clinical tool exists to monitor SNVs simultaneously. Here, an encodable multiplex microsphere-phase amplification (MMPA) sensing platform that combines primer-coded microsphere technology with dual fluorescence decoding strategy to detect SARS-CoV-2 RNA and simultaneously identify 10 key SNVs in the RBD. MMPA limits the amplification refractory mutation system PCR (ARMS-PCR) reaction for specific target sequence to the surface of a microsphere with specific fluorescence coding. This effectively solves the problem of non-specific amplification among primers and probes in multiplex PCR. For signal detection, specific fluorescence codes inside microspheres are used to determine the corresponding relationship between the microspheres and the SNV sites, while the report probes hybridized with PCR products are used to detect the microsphere amplification intensity. The MMPA platform offers a lower SARS-CoV-2 RNA detection limit of 28 copies/reaction, the ability to detect a respiratory pathogen panel without cross-reactivity, and a SNV analysis accuracy level comparable to that of sequencing. Moreover, this super-multiple parallel SNVs detection method enables a timely updating of the panel of detected SNVs that accompanies changing VOCs, and presents a clinical availability that traditional sequencing methods do not.