Positive Plasmid Research Articles

CRISPR/Cas13a-mediated visual detection: A rapid and robust method for early detection of Nosema bombycis in silkworms

The sericulture industry faces a significant threat from the Pebrine disease of silkworms, caused by Nosema bombycis. Nonetheless, the current microscopic diagnostic methods can be time-consuming, labor-intensive, and lacking sensitivity and accuracy. Therefore, it is crucial to develop a novel detection approach that is efficient, highly sensitive, and low-cost. In this regard, the CRISPR/Cas system has the potential to be a fast, accurate, and highly specific method of detection. Herein, using a microplate reader, a portable fluorescence detection device, and test strips as signal output tools respectively, we have efficiently developed three rapid and facile visual detection methods for N. bombycis using a CRISPR/Cas13a system with conjugation of Recombinase polymerase amplification (RPA). We evaluated the sensitivity of this combined technology by comparing it with the positive plasmid standard and the genome standard of N. bombycis. Remarkably, the sensitivity of the CRISPR/Cas13a system for N. bombycis positive plasmid standard based on the microplate reader, portable fluorescence detection device, and test strips was 1 copy/μL, 10 copies/μL, and 1 copy/μL, respectively, while for the N. bombycis genome standards, the detection sensitivity was 10 fg/μL, 10 fg/μL, and 1 fg/μL, respectively. In addition, extensive evaluations have demonstrated that the established technology can accurately detect N. bombycis without cross-reactivity with other pathogens, ensuring a specificity rate of 100%. In brief, this study will provide a practical, efficient, and affordable method for early and rapid detection of N. bombycis in various settings.

Novel Approach for Detection of BRAF and KRAS Mutations Using ARMS (amplification refractory mutation system) Primers for Colon Cancer Therapeutic Response Prediction

Abstract Introduction/Objective KRAS and BRAF V600E mutations are confirmed predictive biomarkers for anti-EGFR monoclonal antibody therapy response and prognosis for colorectal cancer and often considered mutually exclusive with few exceptions (0.05% of mCRC cases). We have developed a multiplex PCR assay utilizing ARMS (amplification refractory mutation system) Primers and Probes for screening and further testing of BRAF mutation status in a 2 step process. Methods/Case Report The Multiplex PCR Assay or BRAF/KRAS mutation was designed as a 5 color assay. Primers and Probes consisted of Wild Type Kras and wild and mutant (V600E/V600K) type BRAF primers and probes. For specimen, Positive and negative control standards made of engineered positive control plasmids for wild type KRAS & BRAF and mutant type V600E & V600K BRAF as well as internal control of bet actin and 2 Patient samples with known BRAF V600E mutant status were run. Mutant type V600E & V600K BRAF were mixed with wild type V600 controls in different ratios as well. Results were interpreted as BRAF wild KRAS wild/ BRAF wild KRAS mutant/ BRAF V600E KRAS wild/BRAF V600K KRAS wild type and NGS was recommended in cases of concurrent BRAF and KRAS mutations. Results (if a Case Study enter NA) Positive/negative/internal control standards for wild type KRAS & BRAF and mutant type V600E & V600K BRAF showed expected results in both CFX 96 and ABI 7500 runs in triplicates. Graphs were able to determine separate Ct values and sigmoidal amplification plots that readily differentiated KRAS wild/BRAF wild/ BRAF V600E types. Mutant type V600E & V600K BRAF that were mixed with wild type V600 controls in different ratios all showed expected results and were able to differentiate between wild and mutant types with accuracy. Also, the 2 patient samples with known (Sanger sequencing-diagnosed) V600E mutation status showed positive Ct values for KRAs wild and V600E probes and negative CT values for V600 wild/V600K probes. No invalid runs or unexpected control results were observed. Conclusion The ARMS BRAF/KRAS multiplex test is a efficient, accurate 2 step assay. In the patients and positive and negative controls prepared, it was able to reliably diagnose KRAS and BRAF mutation status in a matter of 4 hours running time. Given the fact that this is a cost effective assay that can utilize most commonly used Real time PCR machines this test may be clinically implemented if larger clinical trials utilizing tissue samples from a larger number of colon cancer patients are conducted.

Acetylshikonin reduces the spread of antibiotic resistance via plasmid conjugation

The plasmid-mediated conjugative transfer of antibiotic resistance genes (ARGs) stands out as the primary driver behind the dissemination of antimicrobial resistance (AMR). Developing effective inhibitors that target conjugative transfer represents an efficient strategy for addressing the issue of AMR. Here, we studied the effect of acetylshikonin (ASK), a botanical derivative, on plasmid conjugation. The conjugative transfer of RP4-7 plasmid inter and intra species was notably reduced by ASK. The conjugation process of IncI2 and IncX4 plasmids harboring the mobile colistin resistance gene (mcr-1), IncX4 and IncX3 plasmids containing the carbapenem resistance gene (blaNDM-5), and IncFI and IncFII plasmids possessing the tetracycline resistance gene [tet(X4)] were also reduced by ASK. Importantly, the conjugative transfer frequency of mcr-1 positive IncI2 plasmid in mouse peritoneal conjugation model and gut conjugation model was reduced by ASK. The mechanism investigation showed that ASK disrupt the functionality of the bacterial cell membrane. Furthermore, the proton motive force (PMF) was dissipated. In addition, ASK blocked the electron transmission in bacteria's electron transport chain (ETC) through disturbing the quinone interaction, resulting in an insufficient energy supply for conjugation. Collectively, ASK is a potential conjugative transfer inhibitor, providing novel strategies to prevent the spread of AMR.

Investigating the immunological activity of the Hsp70-P113 fusion protein for Mycoplasma ovipneumoniae detection: a groundbreaking study

BackgroundMycoplasmal pneumonia of sheep and goats (MPSG) is an important infectious disease that threatens sheep and goat production worldwide, and Mycoplasma ovipneumoniae (Movi) is one of the major aetiological agents causing MPSG. The aim of this study was to investigate the immunological activity of the Hsp70‒P113 fusion protein derived from Movi and to develop a serological assay for the detection of Movi.MethodsThis study involved codon optimization of the dominant antigenic regions of Movi heat shock protein 70 (Hsp70) and adhesin P113. Afterwards, the optimized sequences were inserted into the prokaryotic expression vector pET-30a( +) through tandem linking with the aid of a linker. Once a positive recombinant plasmid (pET-30a-rHsp70-P113) was successfully generated, the expression conditions were further refined. The resulting double gene fusion target protein (rHsp70‒P113) was subsequently purified using ProteinIso® Ni–NTA resin, and the reactivity of the protein was confirmed via SDS‒PAGE and Western blot analysis. An indirect enzyme-linked immunosorbent assay (i-ELISA) technique was developed to detect Movi utilizing the fusion protein as the coating antigen. The specificity, sensitivity, and reproducibility of all methods were assessed after each reaction parameter was optimized.ResultsThe resulting rHsp70-P113 protein had a molecular weight of approximately 51 kDa and was predominantly expressed in the supernatant. Western blot analysis demonstrated its favourable reactivity. The optimal parameters for the i-ELISA technique were as follows: the rHsp70-P113 protein was encapsulated at a concentration of 5 μg/mL; the serum was diluted at a ratio of 1:50; the HRP-labelled donkey anti-goat IgG was diluted at a ratio of 1:6,000. The results of the cross-reactivity assays revealed that the i-ELISA was not cross-reactive with other goat-positive sera against Mycoplasma mycodies subsp. capri (Mmc), Mycoplasma capricolum subsp. capripneumoniae (Mccp), Mycoplasma arginini (Marg), orf virus (ORFV) or enzootic nasal tumour virus of goats (ENTV-2). The sensitivity of this method is high, with a maximum dilution of up to 1:640. The results of the intra- and inter-batch replication tests revealed that the coefficients of variation were both less than 10%, indicating excellent reproducibility. The analysis of 108 clinical serum samples via i-ELISA and indirect haemagglutination techniques yielded significant findings. Among these samples, 43 displayed positive results, whereas 65 presented negative results, resulting in a positivity rate of 39.8% for the i-ELISA method. In contrast, the indirect haemagglutination technique identified 20 positive samples and 88 negative samples, resulting in a positivity rate of 18.5%. Moreover, a comparison between the two methods revealed a conformity rate of 78.7%.ConclusionThe results obtained in this study lay the groundwork for advancements in the use of an Movi antibody detection kit, epidemiological inquiry, and subunit vaccines.

Enzyme-Mediated Duplex Exponential Amplification: A New Platform for Rapid Screening of <i>Hylurgus ligniperda</i>

As the world's second largest timber importer, wood demand in China has been growing extremely rapidly, leading to an increase of 163% from 2009 to 2018. The plant quarantine pest <i>H. ligniperda Fabricius, 1787</i> is an invasive species frequently intercepted at ports. <i>H. ligniperda</i> causes damage mainly to pine and spruce. To improve the efficiency of on-site inspection and the efficacy of early detection, tight quarantine in ports, time-effective identification, and a national surveillance program for high-risk invasive bark beetles are in urgent need. In this study, a simple, fast and accurate classification method for <i>H. ligniperda </i>is established based on the enzyme-mediated duplex exponential amplification (EmDEA) technique. Partial region from <i>inhibitor of apoptosis 2</i> (<I>IAP2)</I> gene was selected as the target and 6 primer/probe combinations were designed. Through selection, the combination of 3-HY-F3, 3-HY-R2 and 3-HY-RNA5 was chosen as the final primer-probe set, as it showed the lowest Ct with highest final fluorescence signal. Method validation and specificity test using 6 other beetle species living on coniferous wood showed that this method is result reliable and specific. Through parameter analysis with positive plasmid, the detection limit was calculated to be 13.6 copies/μL (9×10<sup>-7</sup> ng DNA/ reaction), much higher than conventional molecular methods such as PCR. The whole process including isothermal amplification, data analysis, and result output can be finished in 30 min, which is highly time-effective. Besides, the operation is simple and little training is needed for non-professionals. The application prospects of this rapid screening system include customs screening in ports, wild survey in non-lab situations and early warning system development. The new analysis platform EmDEA, can also be implemented in rapid detection and identification of other forestry pests.

Plasmid-mediated azithromycin resistance in non-typhoidal Salmonella recovered from human infections.

Mechanisms of non-typhoidal Salmonella (NTS) resistance to azithromycin have rarely been reported. Here we investigate the epidemiology and genetic features of 10 azithromycin-resistant NTS isolates. A total of 457 NTS isolates were collected from a tertiary hospital in Guangzhou. We performed antimicrobial susceptibility tests, conjugation experiments, efflux pump expression tests, whole-genome sequencing and bioinformatics analysis to conduct the study. The results showed that 10 NTS isolates (2.8%) were resistant to azithromycin with minimum inhibitory concentration values ranging from 128 to 512 mg/L and exhibited multidrug resistance. The phylogenetic tree revealed that 5 S. London isolates (AR1-AR5) recognized at different times and departments were closely related [3-74 single-nucleotide polymorphisms (SNPs)] and 2 S. Typhimurium isolates (AR7 and AR8) were clones (<3 SNPs) at 3-month intervals. The azithromycin resistance was conferred by mph(A) gene found on different plasmids, including IncFIB, IncHI2, InFII, IncC and IncI plasmids. Among them, IncFIB, InFII and IncHI2 plasmids carried different IS26-class 1 integron (intI1) arrangement patterns that mediated multidrug resistance transmission. Conjugative IncC plasmid encoded resistance to ciprofloxacin, ceftriaxone and azithromycin. Furthermore, phylogenetic analysis demonstrated that mph(A)-positive plasmids closely related to 10 plasmids in this study were mainly discovered from NTS, Escherichia coli, Klebsiella pneumonia and Enterobacter hormaechei. The genetic environment of mph(A) in 10 NTS isolates was IS26-mph(A)-mrx(A)-mphR(A)-IS6100/IS26 that co-arranged with intI1 harbour multidrug-resistant (MDR) gene cassettes on diverse plasmids. These findings highlighted that the dissemination of these plasmids carrying mph(A) and various intI1 MDR gene cassettes would seriously restrict the availability of essential antimicrobial agents for treating NTS infections.

Hybrid Sequencing-Based Genomic Analysis of Klebsiella pneumoniae from Urinary Tract Infections Among Inpatients at a Tertiary Hospital in Beijing.

Urinary tract infection (UTI) associated with Klebsiella pneumoniae poses a serious threat for inpatients. This study aimed to describe the genomic characteristics of K. pneumoniae causing UTI in a tertiary-care hospital in Beijing, China. A total of 20 K. pneumoniae strains collected from 2020 to 2021 were performed whole-genome sequencing. The Antibiotic susceptibility of 19 common antimicrobial agents was tested against all strains. The multi-locus sequence types (MLSTs) and serotypes were determined from the WGS data. De novo assemblies were used to identify resistance and virulence genes. The presence and characteristics of the plasmids were detected using hybrid assembly of long and short-read data. These K. pneumoniae strains were clustered into nine sequence types (STs) and twelve K-serotypes. All the carbapenem-resistant K. pneumoniae (CRKP) strains acquired carbapenemase blaKPC-2 (n=7). Two CRKP strains exhibited increased resistance to Polymyxin B with MIC ≥ 4 mg/L due to insertion of an IS5-like sequence in the mgrB gene, and they were also involved in a transmission event in Intensive Care Unit. Long-read assemblies identified many plasmids co-carrying multiple replicons. Acquisition of a new IncM2_1 type blaCTX-M-3 positive plasmid was observed after transfer from ICU to neurovascular surgery by comparing the two strains collected from the same patient. K. pneumoniae is a significant pathogen responsible for urinary tract infections. The ST11-KL47 strain, prevalent at our hospital, exhibits a combination of high drug resistance and hypervirulence. It is imperative to enhance ongoing genomic surveillance of urinary tract infection-causing pathogens.

Characterization of blaNDM-19-producing IncX3 plasmid isolated from carbapenem-resistant Escherichia coli and Klebsiellapneumoniae

The increase in the prevalence of carbapenem-producing Enterobacterales (CPE) is a major threat, with the New Delhi metallo-β-lactamase (NDM) enzyme-producing CPEs being one of the major causative agents of healthcare settings infections. In this study, we characterized an IncX3 plasmid harboring blaNDM-19 in Lebanon, recovered from three Escherichia coli belonging to ST167 and one Klebsiella pneumoniae belonging to ST16 isolated from a clinical setting. Plasmid analysis using PBRT, Plasmid Finder, and PlasmidSPAdes showed that all four isolates carried a conjugative 47-kb plasmid having blaNDM-19, and was designated as pLAU-NDM19. We constructed a sequence-based maximum likelihood phylogenetic tree and compared pLAU-NDM19 to other representative IncX3 plasmids carrying NDM-variants and showed that it was closely linked to NDM-19 positive IncX3 plasmid from K. pneumoniae reported in China. Our findings also revealed the route mediating resistance transmission, the IncX3 dissemination among Enterobacterales, and the NDM-19 genetic environment. We showed that mobile elements contributed to the variability of IncX3 genomic environment and highlighted that clonal dissemination in healthcare settings facilitated the spread of resistance determinants. Antimicrobial stewardship programs implemented in hospitals should be coupled with genomic surveillance to better understand the mechanisms mediating the mobilization of resistance determinants among nosocomial pathogens and their subsequent clonal dissemination.

A novel ABC family protein participates in the regulation of fitness cost caused by tet(X4)-bearing plasmids in Escherichia coli

The worldwide prevalence of the plasmid-mediated tigecycline resistance gene tet(X4) in diverse bacterial species challenges the clinical application of tigecycline and poses an increasing threat to global health. However, the underlying mechanisms by which tet(X4)-positive plasmids adapt to different host bacteria and maintain resistance genes remain elusive. Herein, we performed a systematic investigation of the fitness costs of tet(X4)-bearing plasmids in different host strains. We found that the combination of tet(X4) gene and Escherichia coli (E. coli) showed a minimal fitness cost. Meanwhile, E. coli harboring tet(X4)-bearing IncFII plasmid pC41 displayed high lethality in a Galleria mellonella (G. mellonella) infection model, superior conjugation frequency and great competitiveness in murine models. Furthermore, we investigated the regulatory mechanisms for controlling the fitness cost of tet(X4)-bearing pC41 in E. coli. A putative ABC transport family protein, designated as AtlR, balanced the tet(X4) expression and bacterial fitness cost by suppressing the plasmid copy number. It inhibited the plasmid replication by downregulating the expression of repA. Overall, these findings uncover a novel adaptative mechanism accounting for the persistence of tet(X4)-bearing plasmids.

Rapid and sensitive point-of-care diagnosis of human cytomegalovirus infection using RPA-CRISPR technology

BackgroundHuman cytomegalovirus (HCMV) is a common herpesvirus that can cause a range of symptoms, from mild conditions such as fevers to severe illnesses like pneumonia. Early and accurate diagnosis of HCMV infection is crucial, particularly for vulnerable populations with limited medical care. However, current diagnostic methods are often expensive, time-consuming, and require skilled technicians. Materials and methodsWe developed an HCMV-RPA-CRISPR diagnosis platform for the rapid and cost-effective detection of HCMV infection. This method utilizes recombinase polymerase amplification (RPA) to amplify the HCMV target gene isothermally without the need for thermal cycling equipment. The platform integrates the CRISPR/Cas12a system, significantly enhancing specificity and sensitivity. A total of 13 clinical blood samples were tested to evaluate the platform's effectiveness and accuracy. Additionally, a lateral flow assay (LFA) and fluorescence detection were incorporated for straightforward and rapid visual interpretation of the results. ResultsThe assay effectively detected concentrations as low as a single copy of the positive control plasmid per microliter in under 1 h, without requiring specialized equipment or training. In clinical sample evaluations, both the fluorescence readout and LFA exhibited 100% sensitivity and specificity, identifying four HCMV-positive and nine HCMV-negative samples. ConclusionThe HCMV-RPA-CRISPR diagnosis platform is comparably effective to qPCR for HCMV diagnosis. Its applicability in common clinical laboratories, clinics, and point-of-care settings, particularly in resource-limited environments, makes it a valuable tool for widespread HCMV screening and diagnosis.

Electrochemical genosensor based on RNA-responsive human telomeric G-quadruplex DNA: A proof-of-concept with SARS-CoV-2 RNA

In this report, a facile and label-free electrochemical RNA biosensor is developed by exploiting methylene blue (MB) as an electroactive positive ligand of G-quadruplex. The electrochemical response mechanism of the nucleic acid assay was based on the change in differential pulse voltammetry (DPV) signal of adsorbed MB on the immobilized human telomeric G-quadruplex DNA with a loop that is complementary to the target RNA. Hybridization between synthetic positive control RNA and G-quadruplex DNA probe on the transducer platform rendered a conformational change of G-quadruplex to double-stranded DNA (dsDNA), and increased the redox current of cationic MB π planar ligand at the sensing interface, thereby the electrochemical signal of the MB-adsorbed duplex is proportional to the concentration of target RNA, with SARS-CoV-2 (COVID-19) RNA as the model. Under optimal conditions, the target RNA can be detected in a linear range from 1 zM to 1 μM with a limit of detection (LOD) obtained at 0.59 zM for synthetic target RNA and as low as 1.4 copy number for positive control plasmid. This genosensor exhibited high selectivity towards SARS-CoV-2 RNA over other RNA nucleotides, such as SARS-CoV and MERS-CoV. The electrochemical RNA biosensor showed DPV signal, which was proportional to the 2019-nCoV_N_positive control plasmid from 2 to 200000 copies (R2 = 0.978). A good correlation between the genosensor and qRT-PCR gold standard was attained for the detection of SARS-CoV-2 RNA in terms of viral copy number in clinical samples from upper respiratory specimens.

Development, evaluation and application of a polymerase spiral reaction (PSR) based assay on accurate detection of Vibrio parahaemolyticus viable cells and virulence factors from rice product

Vibrio parahaemolyticus is a predominate cause of microbiological food safety. This study aimed to develop a polymerase spiral reaction (PSR) based assay for rapid and visual detection on V. parahaemolyticus. Firstly, the specificity and conservativity of species-specific target tlh gene were determined. Secondly, targeting tlh and virulence factors tdh and trh, PSR assay was developed and optimized using constructed positive plasmid controls and standard strain. Visualized result determination using fluorescent dye was applied to avoid false positive issues. Thirdly, 44 strains were used for specificity evaluation. Fourthly, pure and mixed species artificial contamination systems were included to test its applicability. In addition, a propidium monoazide (PMA)-PSR assay was set up to determine viable but non-culturable (VBNC) cells. Target tlh gene acquired high specificity and PSR assay was optimized to carry out at 65ᵒC for 1 h. Detection limit of PSR assay in purified genomic DNA, plasmid and artificially contaminated food samples were 5.31–53.1 pg/μL, 100 copies, and 103 CFU/mL, respectively, demonstrating the sensitivity as 100-fold higher than regular PCR. With PMA-PSR assay, VBNC cells were accurately determined without the disruption of dead cells and food compositions. Thus, PSR is applicable in detection of V. parahaemolyticus viable cells and virulence factors.

Chitosan oligosaccharide accelerates the dissemination of antibiotic resistance genes through promoting conjugative plasmid transfer

The dissemination of antibiotic resistance genes (ARGs), especially via plasmid-mediated horizontal gene transfer, poses a pervasive threat to global health. Chitosan-oligosaccharide (COS) is extensively utilized in medicine, plant and animal husbandry. However, their impact on microflora implies the potential to exert selective pressure on plasmid transfer. To explore the role of COS in facilitating the dissemination of ARGs via plasmid conjugation, we established in vitro mating models. The addition of COS to conjugation mixtures significantly enhanced the transfer of RP4 plasmid and mcr-1 positive IncX4 plasmid in both intra- and inter-specific. Phenotypic and transcriptome analysis revealed that COS enhanced intercellular contact by neutralizing cell surface charge and increasing cell surface hydrophobicity. Additionally, COS increased membrane permeability by inhibiting the Tol-Pal system, thereby facilitating plasmid conjugative transfer. Furthermore, COS served as the carbon source and was metabolized by E. coli, providing energy for plasmid conjugation through regulating the expression of ATPase and global repressor factor-related genes in RP4 plasmid. Overall, these findings improve our awareness of the potential risks associated with the presence of COS and the spread of bacterial antibiotic resistance, emphasizing the need to establish guidelines for the prudent use of COS and its discharge into the environment.

Successful spread of mcr-1-bearing IncX4 plasmids is associated with variant in replication protein of IncX4 plasmids

IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2, respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1. The copy number of pir-2 encoding plasmids (3.15 ± 0.9 copies) are higher than that of pir-1 encoding plasmids (0.85 ± 0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2, the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared with mcr-1-positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2. Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.

Simplified extraction protocol of citrus tissues for rapid detection of ‘Candidatus Liberibacter asiaticus’ using isothermal recombinase polymerase amplification assay and its application for prevalence studies

Huanglongbing disease caused by ‘Candidatus Liberibacter asiaticus’ is a serious threat to citrus production at global level. Its management largely relies on accurate detection and taking preventive measures to check its spread through sensitive and reliable diagnosis. The available standard nucleic acid-based detection assays rely on use of purified DNA as template and are cumbersome. This study reports a simplified extraction protocol of citrus phloem tissues for rapid recombinase polymerase amplification (RPA) assay-based detection of ‘Candidatus Liberibacter asiaticus’ (CLas)-associated with huanglongbing (HLB). Simplified crude plant extract prepared in phosphate buffer and NaOH:EDTA (using mortar pestle or extraction bag without the use of liquid nitrogen) was successfully employed in the RPA assay. Developed assay targeted specific outer membrane protein (OMP) and ribosomal protein (RP) genomic regions of CLas and could detect its infection up to 10−9 to 10−11 dilution of plant extract and up to 1−10 ag µl−1 of DNA of CLas positive citrus tissues and plasmid DNA having respective gene insert, thus exhibiting sufficient sensitivity. Assay was highly specific and showed no cross-reactivity with the common pathogens infecting citrus exhibiting high reliability. Developed RPA assay was validated using 447 citrus samples collected across the Indian citrus belts, where 79.19 % were detected CLas positive and was more sensitive as compared to PCR wherein 0.45 % to 3.80 % samples were detected false negative. The developed RPA assay rapidly detected CLas infection in simply prepared crude plant extract in cost-effective manner and can be performed at low isothermal temperature in resource-poor laboratories for prevalence studies, bud wood certification and production of CLas-free planting materials of citrus.

Detection of Multiple HPV Types in Liquid Biopsies of Cervical Neoplasia.

More than 95% of cervical cancers and their precancerous lesions are caused by human papillomavirus (HPV). Cell-free (cf) HPV DNA detection in blood samples may serve as a monitoring tool for cervical cancer. In our methodological study, an HPV panel for simultaneous detection of 24 types using mass spectrometry-based analysis was developed for liquid biopsy approaches and tested on HPV positive cell lines, plasmid controls, and cervical high-grade squamous intraepithelial lesions (HSIL) in positive smear samples (n = 52). It was validated in cfDNA blood samples (n = 40) of cervical cancer patients. The HPV panel showed proficient results in cell lines and viral plasmids with a limit of detection of 1 IU (international units)/µL for HPV16/18 and 10GE/µL for HPV11/31/33/39/45/51/52/58/59 and a specificity of 100% for the tested HPV types. In cervical smear samples, HPV DNA was detected with a sensitivity of 98.14%. The overall agreement between the new HPV panel and clinical records was 97.2% (κ = 0.84). In cervical cancer cfDNA, 26/40 (65.0%) tested positive for any HPV type, with most infections due to hrHPV (24/26). HPV positive samples were found in all FIGO stages, with the highest positivity ratio in FIGO III and IV. Even the lowest stage, FIGO I, had 12/23 (52.2%) patients with a positive HPV plasma status. This proof-of-concept paper shows that the described assay produces reliable results for detecting HPV types in a multiplex mass spectrometry-based assay in cervical smear and cfDNA with high specificity and sensitivity in both cohorts. The assay shows potential for liquid biopsy-based applications in monitoring cervical cancer progression.

Development and validation of an insulated isothermal polymerase chain reaction assay for the rapid detection of Mycoplasma synoviae.

Mycoplasma synoviae, which causes the disease known as chicken synovitis, causes serious immunosuppression. We developed a rapid insulated isothermal polymerase chain reaction (iiPCR) assay for on-site detection of M. synoviae using a primer and probe set targeting the variable lipoprotein and haemagglutinin (vlhA) gene. In addition, the specificity, sensitivity, repeatability, and clinical detection of this method were evaluated. Our iiPCR assay detected M. synoviae clinical isolates and samples successfully and produced negative results on Mycoplasma galliscepticum, avian viral arthritis, Escherichia coli, Salmonella, Staphylococcus aureus and Corynebacterium, indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected the prepared positive standard plasmid diluted 10 times (1.00 × 10-1 - 1.00 × 10-10) as a template. The undiluted positive plasmid was positive and double distilled water was negative indicating that the PCR reactions were sensitive, respectively. Finally, the vlhA positive standard plasmid with dilution multiple of 1.00 × 10-4 - 1.00 × 10-6 was repeatedly detected three times to evaluate the repeatability of the iiPCR method established in this experiment showing that the iiPCR of M. synoviae is repeatable. The established iiPCR was also used to detect 50 chicken joint enlargement samples. The thermostatic detection PCR established in this experiment was comparable to a reference real-time PCR (qPCR).

Comparison of quantitative PCR and digital PCR assays for quantitative detection of infectious bronchitis virus (IBV) genome

The quantitative polymerase chain reaction (qPCR) technique is an extensively used molecular tool for the detection and quantification of viral genome load. However, since the qPCR assay is a relative quantification method that relies on an external calibration curve it has a lower assay precision and sensitivity. The digital PCR (dPCR) technique is a good alternative to the qPCR assay as it offers highly precise and direct quantification of viral genome load in samples. In this study, performance characteristics such as the quantification range, sensitivity, precision, and specificity of the dPCR technique was compared to qPCR technique for the detection and quantification of IBV genome loads in serial dilutions of IBV positive plasmid DNA, and IBV infected chicken tissue and swab samples. The quantification range of the qPCR assay was wider than that of the dPCR assay, however dPCR had a higher sensitivity compared to qPCR. The precision of quantification of DNA in plasmid samples in terms of repeatability and reproducibility of results was higher when using the dPCR assay compared to qPCR assay. The quantification results of IBV genome load in infected samples by the qPCR and dPCR assays displayed a high correlation. Hence, our findings suggest that dPCR could be used in avian virology research for improved precision and sensitivity in detection and quantification of viral genome loads.

A Comprehensive Comparison of Rapid RNA Extraction Methods for Detection of SARS-CoV-2 as the Infectious Agent of the Upper Respiratory Tract using Direct RT-LAMP Assay.

The current COVID-19 pandemic has highlighted the need for faster and more cost-effective diagnostic methods. The RNA extraction step in current diagnostic methods, such as real-time qPCR, increases the cost and time required for testing. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) is a promising technique for developing diagnostic tests with desired sensitivity and specificity without the need for RNA extraction. An RT-LAMP assay was developed to detect SARS-CoV-2 with a sensitivity of 0.5 copies of positive control plasmid per microliter in 40 min. Several rapid RNA extraction protocols were evaluated using different reagents, including bovine serum albumin, Triton X-100, Tween 20, proteinase K, guanidine hydrochloride, guanidinium isothiocyanate (GITC), and thermal treatment. Finally, the sensitivity and specificity of the developed direct RT-LAMP were determined using 150 upper respiratory tract samples. Method 10 was selected as the most efficient protocol for the RNA extraction step. The sensitivity and specificity of the developed direct RT-LAMP assay with clinical samples were estimated at 98.4% and 88.8%, respectively. These results suggest that the combination of GITC and Triton X-100 detergent is a highly efficient method for RNA extraction and direct RT-LAMP detection of SARS-CoV-2 in clinical samples, providing a valuable tool for the rapid and cost-effective diagnosis of COVID-19.

Development of a Real-Time qPCR Method for the Clinical Sample Detection of Capripox Virus.

Capripox viruses (CaPVs), including sheep pox virus (SPV), goat pox virus (GPV), and lumpy skin disease virus (LSDV), are the cause of sheep pox (SPP), goat pox (GTP), and lumpy skin disease (LSD) in cattle. These diseases are of great economic significance to farmers, as they are endemic on farms and are a major constraint to international trade in livestock and their products. Capripoxvirus (CaPV) infections produce similar symptoms in sheep and goats, and the three viruses cannot be distinguished serologically. In this study, we developed a real-time quantitative polymerase chain reaction (qPCR) method for identifying CaPV in goats, sheep, and cattle. Clinical samples were tested and verified. The developed assay was highly specific for target viruses, including GPVSPV and LSDV, which had no cross-reaction with other viruses causing similar clinical symptoms. An artificially synthesized positive control plasmid using the CaPV 32 gene inserted into the vector pMD19-T was used as a template, and the correlation coefficient of the linear regression curve (R2) was 0.9916, the estimated amplification efficiency (E) was 96.06%, and the sensitivity (limit of detection, LOD) was 3.80 copies per reaction. Using the clinical samples as a template, the limit of detection (LOD) was 4.91 × 10-5 ng per reaction (1.60 × 10-5-2.13 × 10-3 ng, 95% confidence interval (CI)), which means that this method was one of the most sensitive detection assays for CaPVs. A total of 85 clinical samples from CaPV-infected animals (goats, sheep, and cattle) and 50 clinical samples from healthy animals were used to test and compare the diagnostic results using the Synergy Brands (SYBR) Green-based PCR method recommended by the World Organization of Animal Health (WOAH). Both diagnostic sensitivity (DSe) (95.8-100%, 95% CI) and diagnostic specificity (DSp) (92.9-100%, 95% CI) results of the real-time quantitative PCR (qPCR) and SYBR Green PCR were 100%, and the kappa value (κ) was 1.0 (1-1, 95% CI). In summary, the assay established based on TaqMan probes was advantageous in high specificity, sensitivity, and general applicability and could be a competitive candidate tool for the diagnosis of CaPV in clinically suspected animals.