One-step Assay Research Articles

Novel Electrochemical Sensor Based on One-Step Encapsulation of Metal-Organic Framework (MOF) for Simultaneous Detection of SARS-CoV and SARS-CoV-2.

This study investigates three metal-organic frameworks (MOFs) with distinct Brunauer-Emmett-Teller (BET) surface areas and pore sizes: MOFZr (1274 m2/g, <0.7 nm), MOFAl (371 m2/g, 1.5 nm), and MOFCr (1917 m2/g, 2 nm). Methylene blue (MB, 1.4 × 0.7 × 0.2 nm3) and triferrocene (tri-FC, 1.2 × 0.9 × 0.3 nm3) were adsorbed onto these MOFs. Specific DNA segments targeting SARS-CoV-2 (PR and PO) were employed to block the pores of the MOFs, leading to the formation of DNA-gated MOFs: MOFX/MB/PR and MOFX/tri-FC/PO. These constructs were subsequently integrated with four-arm poly(ethylene glycol) amine (4-arm-PEG-NH2)-modified gold electrodes to create the corresponding sensors (MOFX/MB/PR+MOFX/tri-FC/PO@4-arm-PEG-NH2@Au NPs@GCE). The DNA-gated MOFAl system exhibited the highest loading capacity and a 3-fold increase in sensing efficiency following the introduction of SARS-CoV-2 target DNA, surpassing the performance of the other systems. This study highlights the enhancement of the DNA-gated MOFs when the three-dimensional structures of the guest molecules closely align with the pore sizes of the MOFs. It emphasizes a critical aspect of the traditional design approach for electrochemical sensors based on MOF encapsulation, which often prioritizes BET surface areas while neglecting the compatibility between the sizes of guest molecules and MOF pore diameters. Moreover, the sensor effectively discriminated between SARS-CoV-2 and SARS-CoV by precisely aligning the SARS-CoV-2 target DNA with PR and PO. In contrast, the specific genetic targets (SR) from SARS-CoV showed complete mismatches with PO and a three-base deviation with PR. This differentiation was achieved in a simple one-step assay, even in 5% serum, across a linear concentration range of 10-9 to 10-14 M. This range signifies an expansion of 2 to 3 orders of magnitude compared to sensors that inaccurately selected MOFs.

Highly sensitive time-resolved fluorescent microspheres lateral flow immunoassay for the quantitative detection of triadimefon and its metabolite residues in fruits and vegetables.

Ageneral one-step lateral flow immunochromatographic assay (LFIA) for the quantitative detection of triadimefon (TDF) and triadimenol(TDN) in fruit and vegetable samples was developedusing time-resolved fluorescence microspheres (TRFM) as labels. A specific anti-triadimefon monoclonal antibody (mAb) was conjugated with TRFM to fabricate LFIA test strips. A time-resolved fluorometer as an LFIA reader was applied to obtain quantitative results and assess risk ranges for the LFIA test strips. Under the optimized experimental conditions, the limits of detection (LODs) in buffer/cucumbers/tomatoes/oranges were 0.046ng/mL, 0.135µg/kg, 1.047µg/kg, and 5.811µg/kg, respectively, which are ca. 1000 times lower than that of colloidal gold-labeled strips. The recovery in cucumber/tomato/orange samples was 109.4-116.7%, 87.7-110.9%, and 88.0-111.9%, respectively, indicating that the test strips had good reliability. Coupled with the easily customizable pretreatment procedures for various samples, the LFIA results were obtained within 18min without the need for professional personnel or complicated equipment. TRFM-LFIA for TDF and TDN also shows remarkable specificity and precision. The test strips were also low-cost, portable, and convenientto use. These results indicate the test strips could be utilized as a novel strategy for on-site detection of TDF and TDN, which has the potential to expand and detect other pesticide or insecticide residues in food.

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for In-Field Detection of American Plum Line Pattern Virus.

American plum line pattern virus (APLPV) is the most infrequently reported Ilarvirus infecting stone fruit trees and is of sufficient severity to be classified as an EPPO quarantine A1 pathogen. In late spring, yellow line pattern symptoms were observed on leaves in a few flowering cherries (Prunus serrulata Lindl.) grown in a public garden in Northwest Italy. RNA extracts from twenty flowering cherries were submitted to Ilarvirus multiplex and APLPV-specific RT-PCR assays already reported or developed in this study. One flowering cherry (T22) with mixed prunus necrotic ringspot virus (PNRSV) and prune dwarf virus (PDV) infection also showed infection with APLPV. Blastn analysis of PCR products of the full coat protein (CP) and movement protein (MP) genes obtained from flowering cherry T22 showed 98.23% and 98.34% nucleotide identity with reference APLPV isolate NC_003453.1 from the USA. Then, a LAMP-specific assay was designed to facilitate the fast and low-cost identification of this virus either in the laboratory or directly in the field. The developed assay allowed not only the confirmation of APLPV (PSer22IT isolate) infection in the T22 flowering cherry but also the identification of APLPV in an asymptomatic flowering cherry tree (TL1). The LAMP assay successfully worked with crude flowering cherry extracts, obtained after manually shaking a single plant extract in the ELISA extraction buffer for 3-5 min. The developed rapid, specific and economic LAMP assay was able to detect APLPV using crude plant extracts rather that RNA preparation in less than 20 min, making it suitable for in-field detection. Moreover, the LAMP assay proved to be more sensitive in APLPV detection in flowering cherry compared to the specific one-step RT-PCR assay. The new LAMP assay will permit the estimation of APLPV geographic spread in the territory, paying particular attention to surrounding gardens and propagated flowering cherries in ornamental nurseries.

A-009 Analytical Characterization of a Novel NT-proBNP Assay on a Central Laboratory Platform

Abstract Background N-terminal pro-B-type Natriuretic Peptide (NT-proBNP) or B-type Natriuretic Peptide (BNP) assay(s) are widely measured with a Class 1, Level of Evidence A recommendation in professional heart failure (HF) guidelines for diagnosis and prognosis. We present the analytical characteristics of the first NT-proBNP assay on Beckman Coulter platforms. Methods Measurements were conducted with the Access NT-proBNP assay, a one-step sandwich immunoenzymatic assay using monoclonal capture and detection antibodies, on the DxI 9000 Immunoassay Analyzer with Lithium Heparin (LiHep) plasma samples. Performance was characterized according to CLSI guidelines for: Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ), linearity, imprecision (repeatability, reproducibility) and matrix comparison with LiHep, EDTA and serum. Reference intervals (RI) were evaluated in a healthy, adult U.S. population comprised of 675 subjects with 54.6% females, 79.1% White and 15.1% Black individuals. Exclusion criteria included elevated cardiac troponin, abnormal eGFR and cardiac medications. The 97.5th percentile RI upper limit was defined for age-stratified ranges of &amp;lt;50, 50-75, and &amp;gt;75 years old. Results Performance was LoB=1.1ng/L; LoD=4.8ng/L; LoQ=4.8ng/L; linearity=35,000ng/L. Repeatability ranged from 1.5%-3.5% and reproducibility from 2.3%-7.9% in seven samples ranging from 38ng/L to 23,848ng/L. Matrix measurement regression (n=68) yielded slopes of 1.00, 1.01; intercepts -0.03 to -2.63; r=0.98. Table 1 shows that NT-proBNP values increase with age and women generally have higher values than men. Conclusions The LoB, LoD and LoQ demonstrate highly sensitive low-end characteristics. Repeatability and reproducibility show excellent performance across a wide measurement range. NT-proBNP values are known to be age dependent, and the age-stratified 97.5th percentile RI upper limits were 162 ng/L for &amp;lt;50 years, 311 ng/L for 50-70 years, and 457 ng/L for &amp;gt;75 years. This novel Access NT-proBNP assay offers robust analytical performance as an aid to clinical HF management in acute settings.

Development of a One-Step Multiplex qPCR Assay for Detection of Methicillin and Vancomycin Drug Resistance Genes in Antibiotic-Resistant Bacteria.

The most common antibiotic-resistant bacteria in Korea are methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Pathogen identification in clinical laboratories can be divided into traditional phenotype- and genotype-based methods, both of which are complementary to each other. The genotype-based method using multiplex real-time polymerase chain reaction (PCR) is a rapid and accurate technique that analyzes material at the genetic level by targeting genes simultaneously. Accordingly, we aimed to develop a rapid method for studying the genetic characteristics of antibiotic-resistant bacteria and to provide an experimental guide for the efficient antibiotic resistance gene analysis of mecA detection for MRSA and vanA or vanB detection for VRE using a one-step multiplex qPCR assay at an early stage of infection. As a result, the sensitivity and specificity of the mecA gene for clinical S. aureus isolates, including MRSA and methicillin-susceptible S. aureus, were 97.44% (95% CI, 86.82-99.87%) and 96.15% (95% CI, 87.02-99.32%), respectively. The receiver operating characteristic area under the curve for the diagnosis of MRSA was 0.9798 (*** p < 0.0001). Therefore, the molecular diagnostic method using this newly developed one-step multiplex qPCR assay can provide accurate and rapid results for the treatment of patients with MRSA and VRE infections.

Poly(vinylpyrrolidone)-Enhanced CRISPR-Cas System for Robust Nucleic Acid Diagnostics.

Clustered regularly interspaced short palindromic repeats (CRISPR) technology has opened a new path for molecular diagnostics based on RNA programmed trans-cleavage activity. However, their accessibility for highly sensitive clinical diagnostics remains insufficient. In this study, we systematically investigated the impact of various surfactants on the CRISPR-Cas12a system and found that poly(vinylpyrrolidone) (PVP), a nonionic surfactant, showed the highest enhancement effect among these tested surfactants. Additionally, the enhancement effects of PVP are compatible and versatile to CRISPR-Cas12b and Cas13a systems, improving the sensitivity of these CRISPR-Cas systems toward synthetic targets by 1-2 orders of magnitude. By integrating the PVP-enhanced CRISPR system with isothermal nucleic acid amplification, both the two- and one-step assays exhibited comparable sensitivity and specificity to gold-standard quantitative polymerase chain reaction (qPCR) in the assay of clinical human papillomavirus (HPV) samples, thereby holding significant promise for advancing clinical diagnostics and biomedical research.

The diagnostic performance of the one-step nucleic acid amplification assay for the detection of sentinel lymph node metastases in cytokeratin 19-positive breast cancer: a PRISMA-compliant meta-analysis.

The status of the sentinel lymph nodes (SLNs) is an important prognostic factor for many different types of cancer. The one-step nucleic acid amplification (OSNA) assay has emerged as a rapid intraoperative molecular diagnostic tool for LN metastasis detection. We aimed to evaluate and summarize the value of the OSNA assay for the diagnosis of SLN metastasis in cytokeratin 19 (CK19)-positive breast cancer. To evaluate the diagnostic value, the sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the curve (AUC) were pooled. The threshold effect, followed by subgroup analysis, was performed to explore the source of heterogeneity. A sensitivity analysis was performed to assess the stability of this meta-analysis model. Fagan plots and likelihood ratio scattergrams were used to explore the potential clinical significance. A total of 29 eligible studies, which consisted of 5,331 patients with 10,343 SLNs, were included in this meta-analysis. The pooled sensitivity, specificity, PLR, NLR, and DOR were 0.86 (95% CI: 0.85-0.88), 0.94 (95% CI, 0.94-0.95), 18.00 (95% CI, 13.54-23.92), 0.13 (95% CI, 0.10-0.17), and 138.99 (95% CI, 86.66-222.92), respectively. The AUC was 0.97 (95% CI, 0.95-0.98). Sensitivity analysis showed that four studies had an impact on the pooled results and mainly contributed to the heterogeneity. Fagan's nomogram revealed that the prior probability was 50%, the post-probability positive was 95%, and the post-probability negative was 11%. Our results suggested that OSNA can predict the occurrence of SLN metastasis in CK19-positive breast cancer. However, more well-designed and multicenter diagnostic tests are needed to validate our results.

Enhanced Two-Step LAMP-CRISPR Assay with an Engineered Zst Polymerase for Contamination-Free and Ultrasensitive DNA Detection.

Current loop-mediated isothermal amplification (LAMP)-coupled clustered regularly interspaced short palindromic repeats (LAMP-CRISPR) biosensing in two-step or one-step formats has been applied to next-generation accurate molecular diagnosis. However, two-step LAMP-CRISPR assays intrinsically confront aerosol contamination, while one-step assays possess a compromised detection performance. To this end, we propose an enhanced two-step LAMP-CRISPR assay (ETL-CRISPR) with an engineered Zst polymerase to mediate ultrasensitive DNA detection and thoroughly eliminate aerosol contamination. Instead of supplementing any dTTP, the newly engineered Zst polymerase can efficiently polymerize four oligonucleotides (dATP, dCTP, dGTP, and dUTP), thereby enabling contamination-free and ultrasensitive ETL-CRISPR assay. By targeting the L1 gene of human papillomaviruses (HPV) 16 and the E7 gene of HPV18, our ETL-CRISPR assay achieves high specificity and single-copy level sensitivity within 1 h. Furthermore, we validated the assay by using 85 HPV clinical swab samples with an accuracy of 98.8%, which is comparable to the real-time quantitative polymerase chain reaction. Therefore, ETL-CRISPR provides a straightforward strategy for the contamination-free and ultrasensitive point-of-care diagnosis of clinical pathogens.

ƩS COVID-19 is a rapid high throughput and sensitive one-step quadruplex real-time RT-PCR assay

Real-time reverse transcription polymerase chain reaction (RT-PCR), a standard method recommended for the diagnosis of coronavirus disease 2019 (COVID-19) requires 2–4 h to get the result. Although antigen test kit (ATK) is used for COVID-19 screening within 15–30 min, the drawback is its limited sensitivity. Hence, a rapid one-step quadruplex real-time RT-PCR assay: termed ƩS COVID-19 targeting ORF1ab, ORF3a, and N genes of SARS-CoV-2; and Avocado sunblotch viroid (ASBVd) as an internal control was developed. Based on strategies including designing high melting temperature primers with short amplicons, applying a fast ramp rate, minimizing hold time, and reducing the range between denaturation and annealing/extension temperatures; the assay could be accomplished within 25 min. The limit of detection of ORF1ab, ORF3a, and N genes were 1.835, 1.310, and 1 copy/reaction, respectively. Validation was performed in 205 combined nasopharyngeal and oropharyngeal swabs. The sensitivity, specificity, positive predictive value, and negative predictive value were 92.8%, 100%, 100%, and 97.1%, respectively with 96.7% accuracy. Cohen’s Kappa was 0.93. The newly developed rapid real-time RT-PCR assay was highly sensitive, specific, and fast, making it suitable for use as an alternative method to support laboratory diagnosis of COVID-19 in outpatient and emergency departments.

Molecular characterization of peste des petits ruminants virus and Mycoplasma capricolum subsp. capripneumoniae in small ruminants in northern Mauritania, 2023.

Global eradication of peste des petits ruminants (PPR) is planned for 2030 by international animal health organizations in collaboration with national partners. As the deadline approaches, it is fundamental that the PPR status in each country is determined. In addition, the identification of other pathogens of small ruminants that share common geographical locations and can produce similar clinical signs is also important for differential diagnosis. With this in mind, 37 samples collected from goats and sheep presenting respiratory symptoms in Mauritania in 2023 were screened for the presence of PPR virus, Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies capripneumoniae (Mccp) using a one-step multiplex RT-qPCR assay. None of the samples were positive for Capripoxvirus or P. multocida. Nine of them were positive for PPRV and sequence analysis of a segment of the PPRV nucleoprotein revealed that they belonged to lineage IV and were similar to viruses recently identified in Côte D'Ivoire, Guinea, and Niger indicating transboundary movement. The full genome of one representative virus was also generated. Mccp was identified in eight samples and multi-locus sequence analysis (MLSA) identified them as belonging to MLSA Group 3 together with Mccps identified in China, Tajikistan, Turkey and the United Arab Emirates. This is the first time that such a study has been undertaken in Mauritania and the data generated should be of interest to those involved in the management of goat diseases in Mauritania and neighbouring countries.

Characterizing the bacteriophage PKp-V1 as a potential treatment for ESBL-producing hypervirulent K1 Klebsiella pneumoniae ST258 isolated from veterinary specimens

Background and Aim: The dearth of new antibiotics necessitates alternative approaches for managing infections caused by resistant superbugs. This study aimed to evaluate the lytic potential of the purified bacteriophage PKp-V1 against extended-spectrum β-lactamase (ESBL) harboring hypervirulent Klebsiella pneumoniae (hvKp)-K1 recovered from veterinary specimens. Materials and Methods: A total of 50 samples were collected from various veterinary specimens to isolate K. pneumoniae, followed by antimicrobial susceptibility testing and molecular detection of various virulence and ESBL genes. Multilocus sequence typing of the isolates was performed to identify prevalent sequence types. The bacteriophages were isolated using the double-agar overlay method and characterized using transmission electron microscopy, spot tests, plaque assays, stability tests, and one-step growth curve assays. Results: Among 17 (34%) confirmed K. pneumoniae isolates, 6 (35%) were hvKp, whereas 13 (76%) isolates belonging to the K1 type were positive for the wzy (K1) virulence gene. All (100%) hvKp isolates exhibited the allelic profile of ST258. Overall, PKp-V1 exhibited an 88 % (15/17; (p ≤ 0.05) host range, among which all (100 %; p ≤ 0.01) hvKp isolates were susceptible to PKp-V1. PKp-V1 exhibited a lytic phage titer of 2.4 × 108 plaque forming unit (PFU)/mL at temperatures ranging from 25°C to 37°C. The lytic phage titers of PKp-V1 at pH = 8 and 0.5% chloroform were 2.1 × 108 PFU/mL and 7.2 × 109 PFU/mL, respectively. Conclusion: Although the incidence of ESBL-infected K. pneumoniae in veterinary settings is worrisome, PKp-V1 phages showed considerable lytic action against the host bacterium, indicating the potential of PKp-V1 as a possible alternative therapeutic option against MDR K. pneumoniae. Keywords: antibiotic resistance, bacteriophage, Klebsiella pneumoniae, veterinary.

Advances in plant pathogen detection: integrating recombinase polymerase amplification with CRISPR/Cas systems.

Plant pathogens are causing substantial economic losses and thus became a significant threat to global agriculture. Effective and timely detection methods are prerequisite for combating the damages caused by the plant pathogens. In the realm of plant pathogen detection, the isothermal amplification techniques, e.g., recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have emerged as a fast, precise, and most sensitive alternative to conventional PCR but they often comprise high rates of non-specific amplification and operational complexity. In recent advancements, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease Cas systems, particularly Cas12, have emerged as powerful tools for highly sensitive, specific, and rapid pathogen detection. Exploiting the collateral activities of Cas12, which selectively cleaves single-stranded DNA (ssDNA), novel detection platforms have been developed. The mechanism employs the formation of a triple complex molecule comprising guide RNA, Cas12 enzyme, and the substrate target nucleotide sequence. Upon recognition of the target, Cas12 indiscriminately cleaves the DNA strand, leading to the release of fluorescence from the cleaved ssDNA reporter. Integration of isothermal amplification methods with CRISPR/Cas12 enables one-step detection assays, facilitating rapid pathogen identification within 30min at a single temperature. This integrated RPA-CRISPR/Cas12a approach eliminates the need for RNA extraction and cDNA conversion, allowing direct use of crude plant sap as a template. With an affordable fluorescence visualization system, this portable method achieves 100-fold greater sensitivity than conventional techniques. This review summarizes recent advances in RPA-CRISPR/Cas12a for detecting plant pathogens, covering primer design, field-level portability, and enhanced sensitivity.

One-tube target amplification-free CRISPR-Cas13 autocatalytic system

Clustered regularly interspaced short palindromic repeats (CRISPR) diagnostics have shown great promise for nucleic acid testing. However, CRISPR/Cas13a-based RNA sensing is hindered by the absence of accurate, fast and sensitive assays. Current Cas-based methods generally involve two separate steps: target-amplification and Cas-detection. This two-step method complicates detection, causes cross-contamination, and is difficult to deploy. Therefore, it is necessary to develop a facile one-step Cas13a-based RNA detection assay. We present a one-tube target amplification-free CRISPR-Cas13a autocatalytic circuit system that combines the Cas13a nuclease, T4 polynucleotide kinase, Bsu DNA polymerase and T7 RNA polymerase with a universally designed combinatorial DNA probe to create a one-pot assay for isothermal RNA detection. In this assay, after target RNA recognition, the cis-cleavage and trans-cleavage activities of Cas13 are activated, which leads to the initiation of a Cas13a autocatalytic reaction, resulting in an exponentially amplifying detection signal, thus enabling the detection of approximately 23.01 (95 % CI:16.95-41.23) copies/µL RNA in 45 min. A facile, easy-to-deploy, and one-tube Cas13a-based autocatalytic reaction system was constructed for rapid and highly sensitive RNA detection for the first time. This Cas13a-based autocatalytic system offers not only a versatile platform for directly detecting RNA targets but also a route toward point-of-care nucleic acid testing.

A Finger-Actuated Sample-Dosing Capillary-Driven Microfluidic Device for Loop-Mediated Isothermal Amplification.

Loop-mediated isothermal amplification (LAMP) has attracted significant attention for rapid and accurate point-of-care diagnostics. However, integrating sample introduction, lysis, amplification, and detection steps into an easy-to-use, disposable system has so far been challenging. This has limited the uptake of the technique in practical applications. In this study, we developed a colourimetric one-step LAMP assay that combines thermolysis and LAMP reaction, to detect the SARS-CoV-2 virus in nasopharyngeal swab samples from COVID-19-infected individuals. The limit of detection was 500 copies per reaction at 65 °C for 25 min in reaction tubes. Additionally, we developed a finger-operated capillary-driven microfluidic device with selective PVA coating. This finger-actuated microfluidic device could self-dose the required sample amount for the LAMP reaction and inhibit sample evaporation. Finally, we integrated the LAMP assay into the microfluidic device by short-term pre-storage of the LAMP master mix. Using this device, nasopharyngeal swab samples from COVID-19-infected individuals showed positive results at a reaction time of 35 min at 65 °C. This integrated device may be adapted to detect other RNA viruses of interest rapidly.

Synergistic Antimicrobial Effects of Phage vB_AbaSi_W9 and Antibiotics against Acinetobacter baumannii Infection.

Acinetobacter baumannii is a challenging multidrug-resistant pathogen in healthcare. Phage vB_AbaSi_W9 (GenBank: PP146379.1), identified in our previous study, shows lytic activity against 26 (89.66%) of 29 carbapenem-resistant Acinetobacter baumannii (CRAB) strains with various sequence types (STs). It is a promising candidate for CRAB treatment; however, its lytic efficiency is insufficient for complete bacterial lysis. Therefore, this study aimed to investigate the clinical utility of the phage vB_AbaSi_W9 by identifying antimicrobial agents that show synergistic effects when combined with it. The A. baumannii ATCC17978 strain was used as the host for the phage vB_AbaSi_W9. Adsorption and one-step growth assays of the phage vB_AbaSi_W9 were performed at MOIs of 0.001 and 0.01, respectively. Four clinical strains of CRAB belonging to different sequence types, KBN10P04948 (ST191), LIS2013230 (ST208), KBN10P05982 (ST369), and KBN10P05231 (ST451), were used to investigate phage-antibiotic synergy. Five antibiotics were tested at the following concentration: meropenem (0.25-512 µg/mL); colistin, tigecycline, and rifampicin (0.25-256 µg/mL); and ampicillin/sulbactam (0.25/0.125-512/256 µg/mL). The in vitro synergistic effect of the phage and rifampicin was verified through an in vivo mouse infection model. Phage vB_AbaSi_W9 demonstrated 90% adsorption to host cells in 1 min, a 20 min latent period, and a burst size of 114 PFU/cell. Experiments combining phage vB_AbaSi_W9 with antibiotics demonstrated a pronounced synergistic effect against clinical strains when used with tigecycline and rifampicin. In a mouse model infected with CRAB KBN10P04948 (ST191), the group treated with rifampicin (100 μg/mL) and phage vB_AbaSi_W9 (MOI 1) achieved a 100% survival rate-a significant improvement over the phage-only treatment (8.3% survival rate) or antibiotic-only treatment (25% survival rate) groups. The bacteriophage vB_AbaSi_W9 demonstrated excellent synergy against CRAB strains when combined with tigecycline and rifampicin, suggesting potential candidates for phage-antibiotic combination therapy in treating CRAB infections.

Evaluation and comparison of one-step real-time PCR and one-step RT-LAMP methods for detection of SARS-CoV-2

BackgroundThere is an increasing disease trend for SARS-COV-2, so need a quick and affordable diagnostic method. It should be highly accurate and save costs compared to other methods. The purpose of this research is to achieve these goals.MethodsThis study analyzed 342 samples using TaqMan One-Step RT-qPCR and fast One-Step RT-LAMP (Reverse Transcriptase Loop-Mediated Isothermal Amplification). The One-Step LAMP assay was conducted to assess the sensitivity and specificity.ResultsThe research reported positive samples using two different methods. In the RT-LAMP method, saliva had 92 positive samples (26.9%) and 250 negative samples (73.09%) and nasopharynx had 94 positive samples (27.4%) and 248 negative samples (72.51%). In the RT-qPCR method, saliva had 86 positive samples (25.1%) and 256 negative samples (74.8%) and nasopharynx had 93 positive samples (27.1%) and 249 negative samples (72.8%). The agreement between the two tests in saliva and nasopharynx samples was 93% and 94% respectively, based on Cohen’s kappa coefficient (κ) (P < 0.001). The rate of sensitivity in this technique was reported at a dilution of 1 × 101 and 100% specificity.ConclusionsBased on the results of the study the One-Step LAMP assay has multiple advantages. These include simplicity, cost-effectiveness, high sensitivity, and specificity. The One-Step LAMP assay shows promise as a diagnostic tool. It can help manage disease outbreaks, ensure prompt treatment, and safeguard public health by providing rapid, easy-to-use testing.

A novel and cost-effective real-time RT-PCR targeting 24 nucleotides deletion to differentiate PEDV wild-type and classical attenuated vaccine strains

Porcine Epidemic Diarrhea Virus (PEDV) poses a significant threat to the swine industry, causing severe disease and resulting in substantial economic losses. Despite China’s implementation of a large-scale vaccine immunization strategy in recent years, various strains of PEDV, including classical attenuated vaccine strains, continue to emerge in immunized pig herds. Here, we established a one-step real-time fluorescent reverse transcription PCR (one-step real-time RT-PCR) assay targeting a 24-nucleotide deletion in the ORF1 region of three PEDV classical attenuated vaccine strains, derived from classical strains. This assay effectively distinguishes between PEDV classical attenuated vaccine strains and wild-type strains, and we also explore the causes of this discriminatory target deficiency of this method through phylogenetic and recombination analysis. We found that these three classical attenuated vaccine strains exhibit closer phylogenetic relationships and higher sequence similarity with five cell-adapted strains. Recombination analysis revealed that although recombination is widespread in the PEDV genome, the 24-nucleotide deletion site remains stable without undergoing recombination and can be utilized as a target for identification. Further analysis revealed there are no enzyme cleavage sites near the 24-nucleotide site, suggesting that this deletion may have been lost during the process of culturing these viral strains in cells.The detection method we have established exhibits high specificity and sensitivity to PEDV, without cross-reactivity with other viruses causing diarrheal diseases. A total of 117 swine fecal samples were analyzed using this established one-step real-time reverse transcription PCR assay, indicating the presence of classical attenuated vaccine strains in pig herds in Gansu province, China. Additionally, the designed primer pairs and two probes can be placed in a single reaction tube to differentiate between these two types of strains, effectively reducing detection costs. These findings offer an efficient and cost-effective technological platform for clinical rapid identification testing of both wild-type and classical attenuated vaccine strains of PEDV, as well as for precise investigation of clinical data on natural infections and vaccine immunity in pig herds.

A multiplex one-step fluorescence quantitative differential diagnosis method for severe hand, foot and mouth disease caused by coxsackievirus A16

Hand foot and mouth disease (HFMD) is a common childhood infectious disease which is caused by human enterovirus. The objective of this study was to develop a rapid, sensitive, and accurate method for detecting severe HFMD caused by coxsackievirus A16 (CV-A16). A closed-tube sensitive multiplex one-step reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was applied to detect CV-A16 in the early stage of severe HFMD. This assay targeted the CV-A16 structure protein VP1 to distinguish CV-A16 from other coxsackieviruses The 5’UTR region of enteric viruses was used for detecting the enterovirus and ribonuclease P (RNaseP) was adopted as the internal reference gene. The multiplex MGB probe assay system was used to detect PCR amplicons with different fluorescence reporters in the same system. The limit of detection (LOD) of the RT-qPCR assay for the CV-A16 VP1 gene was 125.893 copies/μl, for the 5′ UTR was 50.1187 copies/μl and for the RNaseP gene was 158.49 copies/μl. Furthermore, specificity analysis showed that the multiplex RT-PCR had no cross-reactivity with the influenza virus, herpangina virus and SARS-COV-2. In correlation analysis, the sensitivity of the multiplex RT-qPCR assay for CV-A16 detection was 100 % (288/288) and the specificity of the multiplex RT-qPCR assay was 99.94 % (3395/3397). The overall agreement between the multiplex RT-qPCR and the results of clinical diagnosis was 99.95 % (3683/3685) and kappa value was 0.996 (p<0.001). The entire procedure, from specimen processing to result reporting, could be completed within 1.5 hours. The one-step multiplex RT-qPCR assay for detecting CV-A16 developed in this study is a good laboratory diagnostic tool for rapid and reliable distinguished detection of CV-A16, especially for severe HFMD patients at an early stage in the disease with low virus load of CV-A16.

Optimization of DAC-ELISA and IC-RT-PCR using the developed polyclonal antibody and one-step RT-PCR assays for detection of Indian citrus ringspot virus in kinnow orange of Punjab, India

Indian citrus ringspot virus (ICRSV), a member of the Mandarivirus genus, causes citrus ringspot disease, impacting kinnow orange quality and yield. Early and accurate detection methods are crucial before visible symptoms manifest in plants. In this study, a 507 bp partial coat protein gene (pCPG) segment was amplified from infected kinnow leaf tissues, cloned into a pET28a vector, and transformed into E. coli BL21(DE3) cells. Induced with IPTG, the cells overexpressed a recombinant partial coat protein (rpCP) of approximately 23 kDa, purified using Ni-NTA resin via affinity chromatography. Validated in western blot with an anti-His antibody, rpCP was used to generate an ICRSV-specific polyclonal antibody (PAb) in rabbits. PAb, optimized at 1:1000 dilution, successfully detected ICRSV in infected kinnow orange leaf extracts via DAC-ELISA and IC-RT-PCR assays. ICRSV was detectable in sample dilutions up to 1:640 and 1:10240 (w/v, g mL−1) by DAC-ELISA and IC-RT-PCR, respectively. One-step RT-PCR assays were also optimized, confirming the presence of ICRSV by amplifying a 507 bp pCPG fragment from total RNA extracted from kinnow orange leaves, with dilution up to 1:5120 (w/v, g mL−1). The result demonstrated that IC-RT-PCR has a 16-fold and 2-fold higher sensitivity than DAC-ELISA and one-step RT-PCR assays.

Simple and fast one-step FRET assay of therapeutic mAb bevacizumab using anti-idiotype DNA aptamer for process analytical technology

We developed an aptamer-based fluorescence resonance energy transfer (FRET) assay capable of recognizing therapeutic monoclonal antibody bevacizumab and rapidly quantifying its concentration with just one mixing step. In this assay, two fluorescent dyes (fluorescein and tetramethylrhodamine) labeled aptamers bind to two Fab regions on bevacizumab, and FRET fluorescence is observed when both dyes come into close proximity. We optimized this assay in three different formats, catering to a wide range of analytical needs. When applied to hybridoma culture samples in practical settings, this assay exhibited a signal response that was concentration-dependent, falling within the range of 50–2000 μg/mL. The coefficients of determination (r2) ranged from 0.998 to 0.999, and bias and precision results were within ±24.0 % and 20.3 %, respectively. Additionally, during thermal and UV stress testing, this assay demonstrated the ability to detect denatured samples in a manner comparable to conventional Size Exclusion Chromatography. Notably, it offers the added advantage of detecting decreases in binding activity without changes in molecular weight. In contrast to many existing process analytical technology tools, this assay not only identifies bevacizumab but also directly measures the quality attributes related to mAb efficacy, such as the binding activity. As a result, this assay holds great potential as a valuable platform for providing highly reliable quality attribute information in real-time. We consider this will make a significant contribution to the worldwide distribution of high-quality therapeutic mAbs in various aspects of antibody manufacturing, including production monitoring, quality control, commercial lot release, and stability testing.