Carryover Contamination Research Articles

A Lateral Flow Biosensor Based on Isothermal Amplification for Visual Identification of Species and Sex from Bloodstain.

The prompt species identification from biological samples at a crime scene can rapidly filter out truly valuable biometric information for subsequent personal identification. Meanwhile, early sex determination can assist in narrowing the pool of suspects. However, the current methods for forensic DNA analysis, particularly in point-of-care scenarios, are often limited by the intricate equipment for signal generation and the laborious procedure for DNA purification. The present study introduces a novel portable lateral flow biosensor that possesses extraction-free and anti-aerosol characteristics for on-site determination of species and sex. The bloodstain can be directly submitted to loop-mediated isothermal amplification (LAMP) for the analysis of both mitochondrial and nuclear DNA. The incorporation of a lateral flow device with gold magnetic nanoparticle probes allows for visual interpretation of results through colorimetric signals while also preventing interference on result judgment from pigments such as hemoglobin. Carryover contamination, which is a disharmonious factor in LAMP, especially as the inherent contradiction derived from uncapping in the lateral flow strategy, has been effectively addressed through the integration of uracil DNA glycosylase without compromising the isothermy throughout the process. As a proof-of-concept experiment, species and sex can be accurately identified within 40 min from trace bloodstains amidst significant background interference by targeting cytochrome b and Y-chromosomal amelogenin. Furthermore, the single-blind study revealed a concordance rate of up to 100% in both simulative degraded and true dated bloodstains. This suggests that this biosensor has the potential to be utilized in forensic DNA analysis at crime scenes.

Heterochronous multiplex real-time PCR with intercalating dye using uracil-DNA N-glycosylase (UNG) and multiple primer pairs to revaluate post PCR product

Real-time PCR with intercalating dyes can only be performed once. The expensive fluorescent hydrolysis probes are target specific and are suitable to detect multiplex targets. Uracil-DNA N-glycosylase (UNG), which specifically hydrolyzes and degrades any uracil-containing PCR products, is often applied before PCR to reduce carryover contamination. We developed an optimized protocol for recovering DNA from PCR products and revaluating by real-time PCR with intercalating dye using UNG processing, which is particularly useful when the sample volume is very small and insufficient for multiple assays of real-time PCR.•A real-time PCR master mix with dUTP instead of dTTP was used.•UNG at 1 % and 10 % concentrations of PCR product volumes were used for the first and second processing.•The second real-time PCR was performed with different primer pairs than the first real-time PCR.

Determination of cinnamaldehyde, thymol and eugenol in essential oils by LC–MS/MS and antibacterial activity of them against bacteria

Plant essential oils contain many secondary metabolites, some of which can effectively inhibit the growth of pathogenic microorganisms, so it is a very promising antibacterial agent. In this study, a qualitative and quantitative method based on high performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) was developed for the simultaneous determination of three bioactive substances, cinnamaldehyde (CNM), thymol (THY), and eugenol (EUG), in the essential oils of plants. Necessary tests for linearity, limit of quantification, recovery, carryover contamination and precision of the method were carried out. Then, the antibacterial activity of 3 bioactive compounds against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was evaluated by minimal inhibitory concentration and the synergistic antimicrobial effect. The results indicated that CNM, THY and EUG had good antibacterial activity. According to the results of fractional inhibitory concentration index (FICI), it is considered that CNM + THY and CNM + THY + EUG has obvious synergistic inhibitory effect on E. coli, and CNM + THY and CNM + EUG has obvious synergistic inhibitory effect on S. aureus. Finally, we analyzed the effect of the bioactive compounds on trace elements in bacteria and found significant changes in magnesium, calcium, copper and iron.

Characterization of Salmonella species from poultry slaughterhouses in South Korea: carry-over transmission of Salmonella Thompson ST292 in slaughtering process.

Salmonella outbreaks linked to poultry meat have been reported continuously worldwide. Therefore, Salmonella contamination of poultry meats in slaughterhouses is one of the critical control points for reducing disease outbreaks in humans. This study examined the carry-over contamination of Salmonella species through the entire slaughtering process in South Korea. From 2018 to 2019, 1,097 samples were collected from the nine slaughterhouses distributed nationwide. One hundred and seventeen isolates of Salmonella species were identified using the invA gene-specific polymerase chain reaction, as described previously. The serotype, phylogeny, and antimicrobial resistance of isolates were examined. Among the 117 isolates, 93 were serotyped into Salmonella Mbandaka (n = 36 isolates, 30.8%), Salmonella Thompson (n = 33, 28.2%), and Salmonella Infantis (n = 24, 20.5%). Interestingly, allelic profiling showed that all S. Mbandaka isolates belonged to the lineage of the sequence type (ST) 413, whereas all S. Thompson isolates were ST292. Moreover, almost all S. Thompson isolates (97.0%, 32/33 isolates) belonging to ST292 were multidrug-resistant and possessed the major virulence genes whose products are required for full virulence. Both serotypes were distributed widely throughout the slaughtering process. Pulsed-field gel electrophoretic analysis demonstrated that seven S. Infantis showed 100% identities in their phylogenetic relatedness, indicating that they were sequentially transmitted along the slaughtering processes. This study provides more evidence of the carry-over transmission of Salmonella species during the slaughtering processes. ST292 S. Thompson is a potential pathogenic clone of Salmonella species possibly associated with foodborne outbreaks in South Korea.

Design of liquid level detection circuit based on sampling probe structure capacitance.

Liquid level detection system is an essential core functional component of automatic clinical medical testing instrument. The conventional liquid level detection method has low detection accuracy and sensitivity, and may have the problem of false detection, which may lead to the inaccurate test results. This paper presents a high sensitivity liquid level detection system based on the principle of variable capacitance. When the sampling probe contacts the liquid level, the probe capacitance will change. The liquid level detection circuit board judges whether the probe contacts the liquid level by sensing the change of probe capacitance. When judging the liquid level signal, the combination of slope detection and amplitude detection is used. The liquid level detection circuit board takes the phase-locked loop(PLL) circuit as the center to detect the change of the capacitance. The reference signal of the PLL is set as a square wave of 375kHz. The double tube probe is used as a part of the tuning capacitor of the voltage controlled oscillator to control the frequency of the output signal, which can realize the rapid phase locking. The experimental results show that the system has accurate detection results, high sensitivity, stable and reliable operation, good dynamic response performance in the case of large and small liquid volume. Compared with other liquid level detection methods based on machine vision, ultrasonic, optics and so on, the proposed liquid level detection system has simpler structure and lower cost, it can avoid the problems of collision, carryover contamination and empty suction by controlling the depth of sampling needle inserted into liquid.

One-pot dual-CRISPR/Cas12b-mediated dUTP-LAMP for rapid and sensitive detection of foodborne pathogens without carryover contamination

Rapid and accurate detection of foodborne pathogens is of great significance for ensuring food safety. Currently, the CRISPR-Dx based on Cas12b, a new and better performing Cas enzyme, is very rare, and the vast majority require a two-step method, facing aerosol contamination. In this work, we reported a one-pot dual-CRISPR/Cas12b mediated dUTP-LAMP method for rapid and sensitive detection of foodborne pathogens without nucleic acid extraction and contamination-free. The design mechanism of sgRNAs in the one-pot CRISPR/Cas12b-mediated LAMP method was investigated in detail for the first time. Moreover, a novel PAM sequence UUN of AapCas12b was discovered, and combined with dUTP-LAMP system for one-pot dual-sgRNA detection without carryover contamination. Our developed method can achieve high specificity, nucleic acid extraction-free detection of 2.33 × 104 CFU/mL Salmonella within 1 h. Furthermore, accurate detection of Salmonella was successfully performed in real samples of milk, fruit and egg, demonstrating the practical significance of the developed method.

Isothermal amplification of long DNA fragments at low temperature by improved strand displacement amplification.

Strand displacement amplification (SDA) is an isothermal amplification technique wherein amplification of a nucleic acid is initiated by nicking enzyme activity at sites flanking the target. Diagnostic SDA is very fast but requires precise optimization and is limited to very short amplicons. Here we report an enhanced approach by addition of single-stranded DNA binding protein, crowding agents and dUTP to enable amplification of kilobase-length products at low temperatures. Additionally, we pair this improved SDA with a novel carryover contamination prevention, eliminating amplifiable DNA at the end of the reaction to reduce contamination risk. Taken together these developments increase the utility and versatility of SDA, broadening the reach of this powerful but uncommonly used method.

Application of bioluminescence assay to assess PCR carryover contamination in forensic DNA laboratories

In forensic DNA testing, PCR-based multilocus short tandem repeat (STR) profiling kits, which have high sensitivity and discriminatory power, are generally used to analyze autosomal and Y-chromosomal DNA profiles. Forensic DNA laboratories require strict quality control for DNA testing, as contamination during analyses leads to incorrect interpretation of DNA profiles. Here, we aimed to apply bioluminescence assay to detect and monitor residual PCR products on laboratory work area and equipment surfaces by targeting dATP in the PCR product and allelic ladder marker. Two commercially available bioluminescence assay kits (CheckLite HS Plus and UltraSnap™) were examined for their sensitivity after confirming their reactivity to dATP. In the assay using CheckLite HS Plus, the lower detectable sample volumes were calculated as 10 pl of PCR product of GlobalFiler and PowerPlex Fusion and 1 pl of PCR product of Yfiler Plus and the allelic ladder marker of GlobalFiler, whereas those in the assay using UltraSnap™ were calculated as 1 nl of PCR product and allelic ladder marker. The sample volumes of these kits were lower than those detected through electrophoresis. Thus, the sensitivity of these kits was sufficient to control PCR carryover contamination in the post-PCR areas. Furthermore, residual PCR products in the post-PCR areas were continuously monitored using a bioluminescence assay. The results showed that the bioluminescence values increased after handling PCR samples for electrophoresis and decreased after decontamination. Therefore, we concluded that the bioluminescence assay is useful for assessing PCR carryover contamination in post-PCR processes in forensic DNA laboratories.

Characterization of Heat-labile Uracil-DNA Glycosylase from Oncorhynchus mykiss and its Application for Carry-over Contamination Control in RT-qPCR.

Heat-labile uracil-DNA glycosylase (HL-UDG) is commonly employed to eliminate carry-over contamination in DNA amplifications. However, the prevailing HL-UDG is markedly inactivated at 50°C, rendering it unsuitable for specific one-step RT-qPCR protocols utilizing reverse transcriptase at an optimal temperature of 42°C. This study aimed to explore novel HL-UDG with lower inactivation temperature and for recombinant expression. The gene encoding an HL-UDG was cloned from the cold-water fish rainbow trout (Oncorhynchus mykiss) and expressed in Escherichia coli with high yield. The thermostability of this enzyme and other enzymatic characteristics were thoroughly examined. The novel HL-UDG was then applied for controlling carry-over contamination in one-step RT-qPCR. This recombinantly expressed truncated HL-UDG of rainbow trout (OmUDG) exhibited high amino acids similarity (84.1% identity) to recombinant Atlantic cod UDG (rcUDG) and was easily denatured at 40°C. The optimal pH of OmUDG was 8.0, and the optimal concentrations of both Na+ and K+ were 10 mM. Since its inactivation temperature was lower than that of rcUDG, the OmUDG could be used to eliminate carry-over contamination in one-step RT-qPCR with moderate reverse transcription temperature. We successfully identified and recombinantly expressed a novel HL-UDG with an inactivation temperature of 40°C. It is suitable for eliminating carry-over contamination in one-step RT-qPCR.

Spatiotemporal dual-dimensional separation enables ultra-simple one-pot CRISPR-based molecular diagnostics

Recently, CRISPR/Cas-based technology has shown great promise for nucleic acid-based molecular diagnostics. However, most established strategies reported two-step operations to achieve high sensitivity but carry the risk of aerosol contamination. Herein, we developed a SpatioTemporal dual-dimensional sepAration-based CRISPR/Cas detection (STAR) system for simple and sensitive one-pot nucleic acid detection, taking advantage of the capillary forces of 3D-printed inner tube which dynamically connects the recombinase polymerase amplification (RPA, inner tube) and CRISPR/Cas12a derived detection (outer tube). With RPA reaction proceeding, the spontaneously defused amplicons will activate the CRISPR/Cas12a reaction, lighting up the inner tube with glamorous fluorescent signals. Benefiting from spatiotemporal dual-dimensional separation, the STAR system not only can address the carryover contamination and incompatibility between two reactions, but also does not require for additional operations once assembled. Our STAR system has exhibited high specificity and sensitivity for the detection of Salmonella typhimurium in both broth and spiked milk samples with detection limits of 100 CFU/mL. With the outstanding performance, the developed STAR system has shown great potential for the development of next-generation point-of-care molecular diagnostics.

Performance Evaluation of Newly-Developed Tumor Markers Assay Kit Based on Flow Fluorescence Assay.

The purpose of this study is to evaluate the performance of recently developed tumor marker clinical kits in China, with the aim of encouraging local medical technology innovation and thus narrowing the research and development gap with foreign kits. The newly established reagent kits were analyzed on the TESMI F3999-Luminex200 flow lattice instrument to verify precision, sensitivity (blank limit), linearity, anti-interference ability, carry-over contamination rate, hook effect, and reference interval verification. Additionally, the newly established reagent kits were compared to other commercially available detection kits (reference reagent kits) to analyze the correlation between the two types of kits. The intra-assay and inter-assay precision had coefficients of variations (CVs) less than 3.50% and 6.91%, respectively. The tumor marker blank limits were lower than the manufacturer's statement. The newly established reagent kits demonstrated excellent linearity (r > 0.99). Rheumatoid factor, triglycerides, bilirubin, and hemoglobin did not have significant interference with the determination of tumor markers. The carry-over contamination rates were all much lower than 3%. At extremely high concentrations of AFP (277,335 ng/mL and 1,031,424 ng/mL), the measured tumor marker values were higher than the upper limit of the linear range and no hook effect occurred. The reference interval was suitable for use in clinical laboratory settings. Correlation analysis indicated a satisfactory relevance and consistency between the newly developed reagent kits and reference reagent kits, with correlation coefficients of r > 0.967 among 654 patients and healthy individuals. The newly developed reagent kits for tumor markers performed well in all evaluated parameters, having the potential for clinical promotion and application.

ElectrochemCap: an integrated detection for loop-mediated isothermal amplification reactions.

Loop-mediated isothermal amplification (LAMP) of genetic materials has emerged as a powerful molecular biology technique with great potential to be a standard point-of-care (POC) technique. This method has found several applications, but it still presents challenges for its direct on-site application, particularly in terms of integrated reaction and detection systems and the risk of carryover contamination. In this work, we propose an innovative solution - an electrochemical microcentrifuge tube cap (ElectrochemCap) based on a screen-printed electrode, a 3D printed adapter and an adhesive layer - which integrates the amplification reaction and its subsequent electrochemical detection in a single device. The design, fabrication, and electrochemical characterization of the ElectrochemCap are reported here, demonstrating its suitability for LAMP detection. Rapidly emerging technologies, such as 3D printing or xurography, are the basis of a prototype that has been validated for the detection of SARS-CoV-2 using reverse transcription LAMP (RT-LAMP), achieving results comparable to those obtained by gold-standard RT-qPCR. Moreover, we have explored the versatility of the ElectrochemCap presenting several additional designs (for containers with different volumes, shapes and materials, as well as multiplexed approaches), expanding its potential applications. Overall, the ElectrochemCap represents an affordable, versatile, and marketable innovation for integrated quantitative electrochemical detection, with enormous possibilities in bioelectroanalytical procedures and portable laboratory setups.

Fully Integrated Microfluidic Platform for Multiplexed Detection of Hunov by a Dynamic Confined-Space-Implemented One-Pot Rpa-Lamp System.

Human norovirus (HuNoV) is the leading cause of nonbacterial acute gastroenteritis, which is highly infectious, rapidly evolving, and easily transmitted through feces. The accurate and early detection of HuNoV subtypes is essential for effective treatment, early surveillance, risk assessment, and disease prevention. In this study, a portable multiplex HuNoV detection platform that combines integrated microfluidics and cascade isothermal amplification, using a streamlined protocol for clinical fecal-based diagnosis is presented. To overcome the problems of carryover contamination and the incompatibility between recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), a Dynamic confined-space-implemented One-pot RPA-LAMP colorimetric detection system (DORLA) is developed by creating a hydrogen bond network. The DORLA system exhibits excellent sensitivity, with detection limits of 10copiesµL-1 and 1copyµL-1 for HuNoV GI and GII, respectively. In addition, a portable diagnostic platform consisting of a thermostatic control module and an integrated 3D-printed microfluidic chip for specific HuNoV capture, nucleic acid pretreatment, and DORLA detection, which enables simultaneous diagnosis of HuNoV GI and GII is developed. A DORLA-based microfluidic platform exhibits satisfactory performance with high sensitivity and portability, and has high potential for the rapid point-of-care detection of HuNoV in clinical fecal samples, particularly in resource-limited settings.

Determination of ertapenem in plasma and ascitic fluid by UHPLC-MS/MS in cirrhotic patients with spontaneous bacterial peritonitis

Abstract Objectives Spontaneous bacterial peritonitis is a frequent severe complication in cirrhotic patients with ascites. Carbapenem antibiotics are currently the treatment of choice for patients with hospital-acquired or healthcare-related infections. However, there is limited evidence available on the efficacy of ertapenem in cirrhotic patients with spontaneous bacterial peritonitis. As a result, the pharmacokynetics and pharmacodynamics of this antibiotic are still unknown. The objective of this study was to develop and validate measurement procedures based on liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to determine ertapenem concentrations in plasma and ascitic fluid. Methods Samples were pretreated by acetronile protein-precipitation. Chromatographic separation is performed on a C18 reversed-phase Acquity®-UPLC®-BEHTM column (2.1 × 100 mm id, 1.7 µm) using a non-linear gradient of water/acetonitrile containing 0.1 % of formic acid at a flow rate of 0.4 mL/min. Ertapenem and its internal standard (ertapenem-D4) are detected by tandem mass spectrometry using positive electrospray ionization and multiple reaction monitoring, and using 476.2 → 346.0/432.2 as mass transition for ertapenem and 480.2 → 350.0 for its internal standard. Results No significant interferences or carry-over contamination were observed. Imprecisions, absolute relative bias, matrix effects and normalized recoveries were ≤14.5 %, ≤9.3 % (92.8–104.5) % and (98.8–105.8) %, respectively. Chromatographic measurement procedures were linear from (0.50–100) mg/L. Conclusions The measurement procedures based on UHPLC-MS/MS developed and validated in this study could be useful in pharmacokynetic and pharmacodynamic studies in subjects with liver cirrhosis who develop spontaneous bacterial peritonitis treated with ertapenem.

Performance evaluation of a novel platelet count parameter, hybrid platelet count, on the BC-780 automated hematology analyzer.

Automated hematology analysis is expected to improve the performance of platelet counting. We evaluated the performance of a new platelet counting, hybrid (PLT-H) and also impedance (PLT-I) and optical (PLT-O) on the BC-780 automated hematology analyzer compared to the international reference method (IRM) in blood samples with thrombocytopenic and platelet interference. The basic platelet count performance of the BC-780 automated hematology analyzer was evaluated according to the requirements of the Clinical Laboratory and Standards Institute (CLSI) Document H26-A2. Additionally, the thrombocytopenic (low PLT count) blood samples and the platelet interference blood samples including fragmented red blood cells (RBCs), microcytes or small RBCs, and giant platelets were determined with the BC-780 hematology analyzer compared to the IRM. Blank counting and the carry-over contamination rate of platelet count using the BC-780 both met the manufacturers' claim. For both 123 thrombocytopenic and 232 platelet interference blood samples (72 fragmented RBCs, 91 microcytes and 51 giant platelets), all three platelet counting methods exhibited high comparability with the IRM (the lowest correlation (r)=0.916). Interestingly, the comparability of PLT-H (r=0.928-0.986) with the IRM was better than that of PLT-I (r=0.916-0.979). The performance of PLT-H in the BC-780 met the manufacturer's specifications. PLT-H exhibits better reproducibility than did PLT-I, correlates well with the PLT-O for thrombocytopenic samples and demonstrates good anti-interference ability. PLT-H counting is therefore recommended as a zero-cost alternative platelet counting method for platelet interference samples in clinical settings.

Nucleic acid based point-of-care diagnostic technology for infectious disease detection using machine learning empowered smartphone-interfaced quantitative colorimetry

We report a nucleic acid-based point of care testing technology for infectious disease detection at resource limited settings by integrating a low-cost portable device with machine learning-empowered quantitative colorimetric analytics that can be interfaced via a smartphone application. We substantiate our proposition by demonstrating the efficacy of this technology in detecting COVID-19 infection from human swab samples, using the RT-LAMP protocol. Comparison with gold standard results from real-time PCR evidences high sensitivity and specificity, ensuring simplicity, portability, and user-friendliness of the technology at the same time. Colorimetric analytics of the reaction output without necessitating the opening of the reaction microchambers enables execution of the complete test workflow without any laboratory control that may otherwise be required stringently for safeguarding against carryover contamination. Seamless sample-to-answer workflow and machine learning-based readout further assures minimal human intervention for the test readout, thus eliminating inevitable inaccuracies stemming from erroneous execution of the test as well as subjectivity in interpreting the outcome. Our results further indicate the possibilities of upgrading the technology to predict the pathogenic load on the infected patients akin to the cyclic threshold value of the real-time PCR, when calibrated with reference to a wide range of ‘training’ data for the machine learner, thereby putting forward the same as viable alternative to the resource-intensive PCR tests that cannot be made readily accessible at underserved community settings.

Accurate molecular identification of different meat adulterations without carryover contaminations on a microarray chip PCR-directed microfluidic lateral flow strip device

Meat adulteration-based food fraud has recently become one of the global major economical, illegal, religious, and public health concerns. In this work, we developed a microarray chip polymerase chain reaction (PCR)-directed microfluidic lateral flow strip (LFS) device that facilitates the accurate and simultaneous identification of beef adulterated with chicken, duck, and pork, especially in processed beef products. To realize this goal, four pairs of amplification primers were designed and applied for specifically amplifying genomic DNA extracted from mixed meat powders in microarray chip. With the prominent advantage of this device lies in the flexible combination and integration of sample loading, detection, and reporting in microstructures, all the DNA amplicons can be individually visualized on the LFS unit, leading to the appearance of test lines (TC line, TD line, TP line, or TB line) as well as the control line (C line) for the species identification and quantification in beef products. Based on this new method, the adulterants were successfully distinguished and identified in mixtures down to 0.01% (wt.%) while the carryover aerogel contamination in routine molecular diagnostic laboratories was effectively avoided. The practicability, accuracy, and reliability of the device were further confirmed by using real-time PCR as a gold standard control on the successful identification of 50 processed ground meat samples sourced from local markets. The method and device proposed herein could be a useful tool for on-site identification of food authentication.

Rapid visual detection of Vibrio parahaemolyticus by combining LAMP-CRISPR/Cas12b with heat-labile uracil-DNA glycosylase to eliminate carry-over contamination.

Vibrio parahaemolyticus is a major pathogen frequently found in seafood. Rapid and accurate detection of this pathogen is important for the control of bacterial foodborne diseases and to ensure food safety. In this study, we established a one-pot system that combines uracil-DNA glycosylase (UDG), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12b (Cas12b) for detecting V. parahaemolyticus in seafood. This detection system can effectively perform identification using a single tube and avoid the risk of carry-over contamination.

A contamination-free and visual method using LAMP (loop-mediated isothermal amplification) and molecular beacon for sensitive and specific detection of rainbow trout (Oncorhynchus mykiss)

Fish species adulteration is a global issue, negatively affecting consumer's rights, public health and marine conservation. LAMP (loop-mediated isothermal amplification) has been widely used for fish species identification, while the commonly-used detection techniques are often time-consuming, prone to carry-over contamination, and sequence-independent. This work selected rainbow trout as a case study, and aimed to develop a novel LAMP assay, combing with a sequence-specific MB (molecular beacon) probe and a simple and portable end-point visual detection device, for rapid detection of rainbow trout with great specificity and superb sensitivity.With the results of the present study, a cost-effective and reliable strategy for the selection of MB's stem-loop structure based on the fluorescence analysis was proved. The molecular beacon LBP-4-1 in the final protocol is characterized by the low background signal and high amplification efficiency. The visual MB-LAMP assay, after optimization, has proved its specificity and applicability, and can detect rainbow trout DNA as little as 0.1 pg in 25 min. Moreover, the risk of cross-contamination can be greatly reduced by the omission of lid-opening, and with the use of a novel end-point visual device, the detection system could be used to screen rainbow trout in the field, without the constraints of complex instruments and other factors. Therefore, the present study offers a simple, fast, accurate method for rainbow trout screening, which could support the identification of authentic aquatic products and help control rainbow trout adulteration.

Rapid and sensitive Cas12a-based one-step nucleic acid detection with ssDNA-modified crRNA

CRISPR-Cas12a RNA-guided complexes have been developed to facilitate the rapid and sensitive detection of nucleic acids. However, they are limited by the complexity of the operation, risk of carry-over contamination, and degradation of CRISPR RNA (crRNA). In this study, a Cas12a-based single-stranded DNA (ssDNA)-modified crRNA (mD-crRNA)-mediated one-step diagnostic method (CasDOS) was established to overcome these drawbacks. mD-crRNA consisted of wild-type crRNA (Wt-crRNA) with ssDNA extensions at the 3′ and 5′ ends. Compared to Wt-crRNA, mD-crRNA exhibited a 100–1000-fold increase in sensitivity in the one-step assay, reducing the cis-cleavage activity of Cas12a to avoid excessive cleavage of the target DNA in the early stages of the reaction, leading to increased amplification and accumulation of the target amplicons, and improved the speed and intensity of the generated fluorescence signal. The detectability of CasDOS was 16.6 aM for the constructed plasmids of Streptococcus agalactiae (GBS), human papillomavirus type 16 (HPV16), and type 18 (HPV18). In clinical trials, CasDOS achieved 100% accuracy in identifying the known genotypes of the five HPV DNA samples. Moreover, CasDOS showed complete concordance with the qPCR results for GBS detection in ten vaginal or cervical swab samples, with a turnaround time from sampling to results within 30 min. In addition, mD-crRNA remained stable after Ribonuclease R treatment, suggesting that it might be more suitable as a raw material for the CRISPR detection kit. In conclusion, we have developed a universal, rapid, and highly sensitive one-step CRISPR detection assay.