Coincidence Rate Research Articles

Development of a Quadruplex RT-qPCR for the Detection of Feline Kobuvirus, Feline Astrovirus, Feline Bufavirus, and Feline Rotavirus

Feline kobuvirus (FeKoV), feline astrovirus (FeAstV), feline bufavirus (FeBuV), and feline rotavirus (FRV) are important pathogens for gastroenteritis, which is characterized by vomiting, diarrhea, and dehydration. Four pairs of primers and probes were designed to target the FeKoV VP1, FeAstV ORF2, FeBuV VP2, and FRV NSP4 genes, and a quadruplex real-time quantitative RT-PCR (RT-qPCR) assay capable of the simultaneous detection of four feline enteroviruses was developed after optimization of reaction conditions. The established quadruplex RT-qPCR assay showed high specificity, sensitivity, and reproducibility. The assay could detect and discriminate FeKoV, FeAstV, FeBuV, and FRV, but not other feline-related pathogens. The limits of detection (LODs) of FeKoV, FeAstV, FeBuV, and FRV were 109.761, 115.834, 125.481, and 113.875 copies/reaction, respectively. The intra- and inter-assay coefficients of variation (CV) were 0.15–1.61% and 0.15–1.59%, respectively. In all, 1869 clinical samples from Guangxi province in Southern China were tested using the developed assay, and the positivity rates of FeKoV, FeAstV, FeBuV, and FRV were 1.93%, 9.36%, 0.32%, and 0.75%, respectively. These samples were also tested using reference assays, and the coincidence rates of the results between the developed and reference methods were 99.63% (FeKoV), 98.72% (FeAstV), 100% (FeBuV), and 100% (FRV), respectively. The results indicated that the developed assay could provide a new detection method for these four viruses associated with feline gastroenteritis.

A novel double antibody sandwich quantitative ELISA for detecting porcine epidemic diarrhea virus infection.

Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0.05 ng/mL and 103.02 50% tissue culture infective dose per mL (TCID50/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92.55% with a kappa value of 0.85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94.29% with a kappa value of 0.88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread.

Winter snowpack loss increases warm-season compound hot-dry extremes

Ongoing warming intensifies snowpack extremes, posing significant hydroclimatic risks to socio-ecological systems. However, the relation between snowpack extremes and subsequent compound hydroclimatic extremes remains unclear. Here, we investigated the impact of snowpack extremes on warm-season compound hydroclimatic extremes in the Northern Hemisphere using multisource datasets from 1980 to 2022. We found widespread increases in deficient, short, and deficient-short snowpack extremes, triggering more compound hot-dry extremes within a month after snowpack disappearance (mean coincidence rate over 0.6, p < 0.05). The impact of compound snowpack extremes exceeded that of individual snowpack extremes in both areas (over 10%) and coincidence rates (over 0.2). Meanwhile, increased intensity, rather than frequency, of snowpack extremes drove mainly the occurrence of compound hydroclimatic extremes. Furthermore, background climate factors, followed by vegetation, topography, and soil, affected relations between snowpack and compound hydroclimatic extremes. These findings will deepen our understanding of the emerging consecutive extremes and improve their predictability.

Does two dimensional templating allow for the use of reduced-size ancillaries in total hip arthroplasty?

Rising costs in healthcare for total hip arthroplasty (THA) mean that new solutions must be considered, such as the use of single-use ancillaries (SUA). The goal of this study was to assess the accuracy of 2D templating in primary THA for the use of reduced-size SUA. Our hypothesis was that the accuracy of 2D templating in primary THA would be higher than 95%, give or take two sizes. This single-centre prospective study included all primary THAs performed over two years. Templating was carried out using 2D templating on anteroposterior pelvic X-rays. The template sizes were compared to the implant sizes. The primary endpoint was the rate of coincidence between digitally templated estimates and the actual implant sizes. The secondary endpoint was the difference of accuracy based on patient parameters. We analysed 512 cases of THA. Accuracy within two sizes was 96.9% for acetabular implants and 98.5% for femoral implants. Accuracy was below the 95% threshold only in patients under 55 and over 85 years old. A BMI above 30.0kg/m2 significantly reduced accuracy but did not fall below the 95% threshold. The operated hip, the type of implant, and the operative indication did not significantly influence templating accuracy. Using reduced-size SUA with five rasps and five reamers depending on template sizes means that THA can be performed in more than 95% of cases allowing the use of compact single use ancillaries.

Detection of Porcine Circovirus (PCV) Using CRISPR-Cas12a/13a Coupled with Isothermal Amplification.

The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness.

Fungibility, accessibility and clinical utility of remote electronic fetal monitoring in improving maternal emotional status compared with traditional method: A multicenter prospective cohort analysis.

Supported by remote signal processing techniques and wireless communication technology, remote electronic fetal monitoring (REFM) has emerged as a promising alternative to traditional electronic fetal monitoring (TEFM) in clinical practice. The aim of this study was to evaluate the comparability, accessibility, and clinical utility of REFM in contrast to TEFM. This was a multicenter prospective cohort study. A cohort of 2900 pregnant women were enrolled from three medical centers between June 1, 2021 and June 31, 2022. Among them, 800 utilized REFM, with 760 of them completing the self-rating anxiety scale (SAS) and self-rating depression scale (SDS) assessments using the devices for 1 month. The control group comprised 2100 pregnant women who did not use REFM. Additionally, 80 pregnant women concurrently employed both REFM and TEFM, and their respective curve coincidence rates were determined through curve fitting. Primary outcomes encompassed pregnancy outcomes in both groups, average curve coincidence rates between REFM and TEFM, as well as SDS and SAS scores. Among the 760 pregnant women who completed SAS and SDS assessments, their average SAS scores before and after 1 month of REFM usage were 43.09 ± 8.04 and 41.58 ± 6.59, respectively. Concurrently, the average SDS scores before and after 1 month of REFM usage were 45.45 ± 9.60 and 44.80 ± 9.17, respectively. A statistically significant decrease was observed in SAS scores (P = 0.005), whereas no significant difference was noted in SDS scores (P = 0.340). Furthermore, a statistically significant difference in the rate of adverse pregnancy outcomes (neonatal asphyxia) emerged between the two groups, those who employed REFM and those who did not (P = 0.021). In the subset of 80 pregnant women employing both REFM and TEFM, all 80 results showed precise congruence between the two methods. The average coincidence rate was determined to be 79.45% ± 12.64%. REFM contributes to improved pregnancy outcomes and exhibits a high level of concordance with TEFM, thereby accurately reflecting the quality of fetal heart monitoring. Additionally, REFM effectively mitigates pregnant women's anxiety. Thus, REFM demonstrates comparability, accessibility, and clinical utility.

Prestack reservoir prediction method in ray parameter domain based on wide azimuth ocean bottom node seismic data

AbstractWide azimuth seismic data play an important role in deep reservoir prediction. According to the Paleogene clastic rock reservoir prediction, the high‐density and wide azimuth 3D seismic data acquisition of ocean bottom nodes was first carried out in a Chinese offshore oilfield in 2019. After high precision amplitude‐preserving processing, we obtained the high‐quality wide azimuth gathers. However, the research on anisotropy and reservoir prediction using wide azimuth seismic data mainly focuses on carbonate and bedrock intervals, which is not suitable for clastic rock reservoir prediction. Therefore, this paper innovatively proposes a clastic rock reservoir prediction method, which studies prestack reservoir prediction in the ray parameter domain based on wide azimuth ocean bottom node seismic data. Based on the azimuth gathers, we can obtain elastic parameters through prestack amplitude versus offset inversion, which is used to characterize the reservoir, so it is significant to obtain high precision elastic parameters in order to get highly reliable reservoir prediction results. In this paper, we develop an amplitude versus offset inversion method based on the Bayesian theory in ray parameter domain, the output of which is density, P‐wave impedance and Vp/Vs. These elastic parameters have high precision, and density data are valuable input for reservoir characterization because they are sensitive to the lithology of clastic rock reservoir at different orientations. In ray parameter domain inversion, the ray path of seismic wave propagation is considered polyline, which is more consistent with the actual situation; thus, extracted amplitudes of P gathers used in inversion are more accurate. In addition, the reflection coefficient formula in ray parameter domain has higher precision when the incident angle is large. The inversion based on the Bayesian theory can improve the stability of the inversion. Test on the actual data shows that the result of ray parameter domain inversion with a Bayesian scheme is more accurate, stable and reliable. Based on the above high precision density inversion results, an innovative wide azimuth data reservoir prediction technology based on elliptical short‐axis fitting was proposed. The actual prediction of the deep reservoir in the Bohai oilfield shows that sand thickness fitting prediction results in the short axis can best match the actual drilling sandstone thickness. The coincidence rate is 86% and the short‐axis fitting results are more in agreement with geological laws. Theoretical research and practical applications have shown that this method is feasible and effective, with high prediction accuracy, computational efficiency and strong application value.

Determination of hepatitis B virus (HBV) DNA in serum by nucleic acid screening

The determination of serum hepatitis B Virus (HBV) DNA is of paramount significance in the comprehensive management of HBV infection. It aids in diagnosis, disease staging, and treatment monitoring, ultimately improving patient outcomes and reducing the burden of HBV-related morbidity and mortality. In this work, serum HBV DNA detection was performed using a novel nucleic acid screening system. The sensitivity, selectivity, repeatability, cross reactivity, and HBV DNA stability were evaluated and compared with published data. The detection limit of 95% was 29.12 IU/mL and the 95% confidence interval was from 23.71 to 34.55 IU/mL. Blood samples that contain free hemoglobin (1 g/L, 2 g/L, 4 g/L) and triglycerides (3 mmol/L, 6 mmol/L and 9 mmol/L) were also analyzed. The results demonstrated that the system can also be used in the presence of free hemoglobin and triglycerides. The mean of the cycle threshold (Ct) in 5-day replicates was 35.71 ± 0.35 with a coefficient of variation (CV) of 0.97%. The results of negative samples in the anti-cross contamination experiment showed no response. The coincidence rate for 3001 samples over 20 days of real-time stability measurements and external quality assessment samples was 100%. The results demonstrate the effectiveness of the nucleic acid system for HBV-DNA, providing a theoretical basis for other small and medium-sized blood stations or hospitals to these devices.

Performance evaluation of four kits for the detection of neutralizing antibody against SARS-CoV-2 in human serum

To evaluate four novel SARS-CoV-2 neutralizing antibody assay kits' application in neutralizing antibodies of population. Questionnaires from the voluntary participating researchers and selected the qualified questionnaires to analyse. For negative and positive coincidence rate, four novel SARS-Cov-2 neutralization antibody assay kits were tested. For within-run and between-run Precision verification study, four serum samples with two high and two low titer neutralizing antibodies were used to analyse. Based on the questionnaires, 175 qualified samples were divided into two groups. (1) negative neutralizing antibodies group: 31 samples had not been infected with the novel SARS-Cov-2 nor received the vaccine within the past one year; (2) positive neutralizing antibodies group: 144 samples were infected by COVID-19. There was 28 negative and 3 positive neutralizing antibodies of the individuals among the 31 negative samples which based on the questionnaires. The negative rates of 28 negative individules tested by GenScript, Vazyme and Hygeianey were 82.14 %, 60.71 % and 17.85 %, while the positive rates of the 147 positive samples were 93.87 %, 95.23 % and 100 %. The within-run coefficient of variations (C·V) of PBNAs, GenScript, Vazyme and Hygeianey were 11.49 %, 9.12 %, 7.97 % and 7.48 %, while the between-run coefficient of variations (C·V) were 21.37 %, 14.21 %, 12.29 % and 11.78 %. Due to the large within-run and between-run coefficient of variations, PBNAs was not suitable for large-scale promotion, while ELISAs could be leveraged for routine monitoring the titer of neutralizing antibodies against SARS-CoV-2.

Analytical and Diagnostic Performance of a Dual-Target Blood Detection Test for Hepatocellular Carcinoma.

Surveillance approaches with high sensitivity and specificity for hepatocellular carcinoma (HCC) are still urgently needed. Previous studies have shown that methylation of GNB4 and Riplet can effectively diagnose HCC. This study plan to analyze the performance of a blood test for detecting HCC using GNB4 and Riplet methylation. This study mainly investigated the analytical performance of the dual-target HCC blood test (DT-HBT), including cut-off value, limit of detection (LOD), precision, analytical specificity, and coincidence rate. In addition, the detection performance for HCC was validated in 1030 clinical plasma samples (214 HCC and 816 non-HCC). Plasma samples from 25 HCC patients after hepatectomy were collected to assess the feasibility of the kit for postoperative recurrence monitoring. All analytical performance of the DT-HBT met prespecified requirements. The LOD for GNB4, Riplet, and β-actin was 1% methylation/100 copies/μL with cut-offs of 43, 43, and 35, respectively. The DT-HBT showed excellent precision, within 5% CV. It had a specificity of 91.5% for detecting other cancers, and 100% for breast, lung, and bladder cancer. No cross-reactions were observed with 9 potential interfering substances. The DT-HBT achieved a 100% coincidence rate in detecting reference and clinical samples. The clinical performance study found that the kit showed a sensitivity of 81.7% for stage I HCC, and an overall sensitivity and specificity of 87.4% and 92.3%, respectively. The detection sensitivity for postoperative recurrent patients was 95.8%, with a specificity of 100%. The analytical performance of the DT-HBT met prespecified criteria. It provided HCC patients with a reliable and high-performing new blood test for the HCC diagnosis and surveillance. ClinicalTrials.gov identifier: NCT05685524.

Recombinant Polymerase Amplification Coupled with CRISPR/Cas12a Detection System for Rapid Visual Detection of Porcine Circovirus 3.

The porcine circovirus type 3 (PCV3) infection is an emerging disease associated with clinical signs of porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs. Currently, there is a lack of effective vaccines and therapeutics against this disease. Therefore, rapid, effective, sensitive, and specific detection methods are crucial for the timely identification, prevention, and control of PCV3. In this study, we developed one- and two-pot visual detection methods for PCV3 using a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a detection system combined with recombinase polymerase amplification (RPA). These two methods demonstrated no cross-reactivity with eight other swine viruses and exhibited minimum detection limits of five and two copies of viral DNA, respectively, revealing their high specificity and sensitivity. During a clinical sample detection within 30 min, the coincidence rates between the one- and two-pot detection methods and real-time quantitative polymerase chain reaction (qPCR) were 100%. In conclusion, both one- and two-pot RPA-CRISPR/Cas12a detection methods have significant potential for the rapid, sensitive, and specific visual detection of PCV3.

Establishment of ELISA method for canine adenovirus type 1.

Canine adenovirus (CAdV) had a high prevalence in fox populations and induced fox encephalitis. No ELISA kits specifically for CAdV-1 antigen had been commercialized for foxes in China. It is crucial to develop a rapid and accurate ELISA method for detecting of CAdV-1. The monoclonal antibodies (mAbs, IgG1A) and HRP-labeled polyclonal antibodies (pAbs) were used to establish the ELISA method in this experiment. The results showed that the optimal concentration and coating time for the mAbs (IgG1A) were 2.15 μg/mL and overnight at 4°C, respectively. The dilution ratio of the HRP-labeled pAbs was 1:2000. Five percent skimmed milk was selected as the blocking agent. The optimal incubation times for blocking, CAdV-1, and HRP-labeled pAbs were all 1 h. The cut-off value for negative rectal swab was determined to be 0.366 ± 0.032. The maximum dilution ratio was 100 TCID50/mL. The ELISA method was positive to CAdV-1, and that was negative to CAdV-2, Canine Parvovirus (CPV) and Canine Distempervirus (CDV). The ELISA method showed good repeatability, sensitivity, and specificity. Compared with RT-PCR, the sensitivity, specificity, and coincidence rates of the ELISA method were 93.75, 90.9, and 92.86%, respectively. These results indicate that the established ELISA method can be used for the large-scale screening and epidemiology surveillance of CAdV-1 in foxes.

Comparative analysis of metagenomic and targeted next-generation sequencing for pathogens diagnosis in bronchoalveolar lavage fluid specimens.

Although the emerging NGS-based assays, metagenomic next-generation sequencing (mNGS) and targeted next-generation sequencing (tNGS), have been extensively utilized for the identification of pathogens in pulmonary infections, there have been limited studies systematically evaluating differences in the efficacy of mNGS and multiplex PCR-based tNGS in bronchoalveolar lavage fluid (BALF) specimens. In this study, 85 suspected infectious BALF specimens were collected. Parallel mNGS and tNGS workflows to each sample were performed; then, we comparatively compared their consistency in detecting pathogens. The differential results for clinically key pathogens were confirmed using PCR. The microbial detection rates of BALF specimens by the mNGS and tNGS workflows were 95.18% (79/83) and 92.77% (77/83), respectively, with no significant difference. mNGS identified 55 different microorganisms, whereas tNGS detected 49 pathogens. The comparative analysis of mNGS and tNGS revealed that 86.75% (72/83) of the specimens were complete or partial concordance. Particularly, mNGS and tNGS differed significantly in detection rates for some of the human herpesviruses only, including Human gammaherpesvirus 4 (P<0.001), Human betaherpesvirus 7 (P<0.001), Human betaherpesvirus 5 (P<0.05) and Human betaherpesvirus 6 (P<0.01), in which tNGS always had higher detection rates. Orthogonal testing of clinically critical pathogens showed a total coincidence rate of 50% for mNGS and PCR, as well as for tNGS and PCR. Overall, the performance of mNGS and multiplex PCR-based tNGS assays was similar for bacteria and fungi, and tNGS may be superior to mNGS for the detection of DNA viruses. No significant differences were seen between the two NGS assays compared to PCR.

Development of a triplex RT-RAA-LFA assay for the rapid differential diagnosis of porcine epidemic diarrhea virus, porcine deltacoronavirus and transmissible gastroenteritis virus

Porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) are three clinically common coronaviruses causing diarrhea in pigs, with indistinguishable clinical signs and pathological changes. Rapid, portable and reliable differential diagnosis of these three pathogens is crucial for the prompt implementation of appropriate control measures. In this study, we developed a triplex nucleic acid assay that combines reverse transcription recombinase-aided amplification (RT-RAA) with lateral flow assay (LFA) by targeting the most conserved genomic region in the ORF1b genes of PEDV, PDCoV and TGEV. The entire detection process of the triplex RT-RAA-LFA assay included 10-min nucleic acid amplification at 42 °C and 5-min visual LFA readout at room temperature. The assay could specifically differentiate PEDV, PDCoV and TGEV without cross-reaction with any other major swine pathogens. Sensitivity analysis showed that the triplex RT-RAA-LFA assay was able to detect the viral RNA extracted from the spiked fecal samples with the minimum of 1 × 100 TCID50 PEDV, 1 × 104 TCID50 PDCoV, and 1 × 102 TCID50 TGEV per reaction, respectively. Further analysis showed that the 95 % detection limit (LOD) of triplex RT-RAA-LFA for PEDV, PDCoV, and TGEV were 22, 478, and 205 copies of recombinant plasmids per reaction, respectively. The diagnostic performance of triplex RT-RAA-LFA was compared with that of PEDV, PDCoV and TGEV respective commercial real-time RT-PCR kits by testing 114 clinical rectal swab samples in parallel. The total diagnostic coincidence rates of triplex RT-RAA-LFA with real-time RT-PCR kits of PEDV, PDCoV and TGEV were 100 %, 99.1 % and 99.1 %, respectively, and their Kappa values were 1.00, 0.958 and 0.936, respectively. Collectively, the RT-RAA-LFA assay is a powerful tool for the rapid, portable, visual, and synchronous differential diagnosis of PEDV, PDCoV, and TGEV.

From Lab to Home: Ultrasensitive Rapid Detection of SARS-CoV-2 with a Cascade CRISPR/Cas13a-Cas12a System Based Lateral Flow Assay.

Currently, CRISPR/Cas-based molecular diagnostic techniques usually rely on the introduction of nucleic acid amplification to improve their sensitivity, which is usually more time-consuming, susceptible to aerosol contamination, and therefore not suitable for at-home molecular testing. In this research, we developed an advanced CRISPR/Cas13a-Cas12a-based lateral flow assay that facilitated the ultrasensitive and rapid detection of SARS-CoV-2 RNA directly from samples, without the need for nucleic acid amplification. This method was called CRISPR LFA enabling at-home RNA testing (CLEAR). CLEAR used a novel cascade mechanism with specially designed probes that fold into hairpin structures, enabling visual detection of SARS-CoV-2 sequences down to 1 aM sensitivity levels. More importantly, CLEAR had a positive coincidence rate of 100% and a negative coincidence rate of 100% for clinical nasopharyngeal swabs from 16 patients. CLEAR was particularly suitable for at-home molecular testing, providing a low-cost, user-friendly solution that can efficiently distinguish between different SARS-CoV-2 variants. CLEAR overcame the common limitations of high sensitivity and potential contamination associated with traditional PCR-based systems, making it a promising tool for widespread public health application, especially in environments with limited access to laboratory resources.

Development and validation of a rapid five-minute nucleic acid extraction method for respiratory viruses

BackgroundThe rapid transmission and high pathogenicity of respiratory viruses significantly impact the health of both children and adults. Extracting and detecting their nucleic acid is crucial for disease prevention and treatment strategies. However, current extraction methods are laborious and time-consuming and show significant variations in nucleic acid content and purity among different kits, affecting detection sensitivity and efficiency. Our aim is to develop a novel method that reduces extraction time, simplifies operational steps, and ensures high-quality acquisition of respiratory viral nucleic acid.MethodsWe extracted respiratory syncytial virus (RSV) nucleic acid using reagents with different components and analyzed cycle threshold (Ct) values via quantitative real-time polymerase chain reaction (qRT-PCR) to optimize and validate the novel lysis and washing solution. The performance of this method was compared against magnetic bead, spin column, and precipitation methods for extracting nucleic acid from various respiratory viruses. The clinical utility of this method was confirmed by comparing it to the standard magnetic bead method for extracting clinical specimens of influenza A virus (IAV).ResultsThe solution, composed of equal parts glycerin and ethanol (50% each), offers an innovative washing approach that achieved comparable efficacy to conventional methods in a single abbreviated cycle. When combined with our A Plus lysis solution, our novel five-minute nucleic acid extraction (FME) method for respiratory viruses yielded superior RNA concentrations and purity compared to traditional methods. FME, when used with a universal automatic nucleic acid extractor, demonstrated similar efficiency as various conventional methods in analyzing diverse concentrations of respiratory viruses. In detecting respiratory specimens from 525 patients suspected of IAV infection, the FME method showed an equivalent detection rate to the standard magnetic bead method, with a total coincidence rate of 95.43% and a kappa statistic of 0.901 (P < 0.001).ConclusionsThe FME developed in this study enables the rapid and efficient extraction of nucleic acid from respiratory samples, laying a crucial foundation for the implementation of expedited molecular diagnosis.

Prenatal Diagnosis of Persistent Left Superior Vena Cava Using High-Definition Flow Render Mode and Spatiotemporal Image Correlation.

This study aimed to assess the use of two-dimensional (2D) ultrasound combined with high-definition flow (HD-flow) render mode and spatiotemporal image correlation (STIC) in diagnosing and classifying fetal persistent left superior vena cava (PLSVC). Overall, 114 cases of fetal PLSVC were diagnosed using 2D ultrasound combined with STIC, and 114 normal fetuses of the same gestational week were selected. These cases were retrospectively analyzed to evaluate the effectiveness of the diagnostic approach. All 114 PLSVC cases were diagnosed using 2D ultrasound combined with STIC. Although the diagnostic coincidence rate of PLSVC in the HD-flow combined with STIC was similar to that in the 2D ultrasound combined with HD-flow (96.8 vs 96.2%), 2D ultrasound with STIC enabled dynamic visualization of the PLSVC, furthering prenatal diagnosis. These cases were classified as type I PLSVC: 80 cases of type Ia, 29 cases of type Ib, and 5 cases of type Ic. Seventy isolated PLSVC cases (61.4%) were noted, whereas 44 cases (35.6%) were associated with concomitant structural abnormalities. Intracardiac structural malformations accounted for the highest proportion (n = 53, 58.89%), followed by single umbilical artery and facial/bodily abnormalities (n = 10, 11.11%). Combining HD-flow and STIC complements 2D ultrasound in diagnosing and classifying fetal PLSVC, demonstrating significant clinical relevance.

Development and Application of Colloidal Gold Test Strips for the Rapid Detection of Canine Brucellosis.

Brucellosis is a global problem, with the causative agent being the genus Brucella. B. canis can cause undulant fever in dogs, which is a zoonotic disease that can spread not only among dogs but also to humans. This poses a public health threat to society. In this study, a rapid and straightforward immune colloidal gold test strip was developed for the diagnosis of canine brucellosis through the detection of anti-LPS antibodies in serum samples. Rabbit anti-canine IgG conjugated with colloidal gold was employed as the colloidal gold-labeled antibody. The extracted high-purity R-LPS was employed as the capture antigen in the test line (T-line), while goat anti-rabbit IgG was utilized as the capture antibody in the control line (C-line). The colloidal gold strip exhibited high specificity in the detection of brucellosis, with no cross-reaction observed with the common clinical canine diseases caused by Canine coronavirus (CCV), Canine distemper virus (CDV), and Canine parvovirus (CPV). In comparison to the commercial iELISA kit, the sensitivity and specificity of the colloidal gold test strip were found to be 95.23% and 98.76%, respectively. The diagnostic coincidence rate was 98.47%. The findings of this study indicate that colloidal gold test strips may be employed as a straightforward, expeditious, sensitive, and specific diagnostic instrument for the identification of canine brucellosis, particularly in resource-limited regions.

Risk factors for pathological upgrading and noncurative resection in patients with gastric mucosal lesions after endoscopic submucosal dissection

BackgroundThe pathological results obtained from endoscopic forceps biopsy (EFB) do not always align with the findings of postoperative endoscopic submucosal dissection (ESD). Furthermore, as ESD becomes more widespread, the number of noncurative endoscopic cases increases; thus, an accurate preoperative diagnosis and an appropriate treatment method are crucial. The purpose of this study was to explore the risk factors for postoperative pathological upgrading and noncurative resection and to gather experience in clinical and pathological diagnosis.MethodsFrom March 2016 to November 2023, 292 ESD specimens were collected from 262 patients with gastric mucosal lesions. Clinicopathological information, the coincidence rate of pathological diagnosis between EFB and ESD specimens, and risk factors related to noncurative resection were analyzed retrospectively.ResultsThe overall upgraded pathological diagnosis rate between EFB and ESD was 26.4%. The independent predictors for the upgraded group included proximal stomach lesions, lesion size > 2 cm, surface ulceration, and surface nodules. Twenty of the 235 early gastric cancer (EGC) patients underwent noncurative ESD resection. Multivariate analysis showed that undifferentiated carcinoma and tumor infiltration into the submucosa were significantly associated with noncurative resection.ConclusionBiopsy cannot fully represent the lesions of gastric intraepithelial neoplasia (GIN). When a suspected epithelial dysplasia is suspected, a careful endoscopic examination should be conducted to evaluate the lesion site, size, and surface characteristics to ensure an accurate diagnosis. Noncurative endoscopic resection is associated with undifferentiated carcinoma and submucosal infiltration. Clinicians must be familiar with these predictive factors for noncurative resection and select the appropriate treatment for their patients.

Multiprobe Amplification (MPA) with Melting Curve Analysis: A Highly Stable and Cost-Effective Platform for the Simultaneous Detection of Eight Potential Bacterial Bioterrorism Agents in Complex Samples.

We aimed to develop an efficient detection platform that can identify a larger number of suspicious samples in a single test, saving time, manpower, and material costs, and providing vital support to the public health system in coping with the current challenging and dynamic bioterrorism threat landscape, particularly in regions of turmoil and conflict. We have successfully developed a high-throughput, multitarget fluorescent array detection platform by effectively combining integrating multiprobe amplification (MPA) with melting curve analysis. Specifically, we have established reliable laboratory testing methods for eight highly pathogenic bacteria, including Bacillus anthracis, Yersinia pestis, Brucella spp., Burkholderia pseudomallei, Francisella tularensis, Vibrio cholerae, Salmonella typhi, and Staphylococcus aureus. Our method achieves sensitive and specific simultaneous detection of eight target bacteria in one well by optimizing the reaction conditions of MPA. In the assessment of 192 simulated environmental samples, both positive and negative coincidence rates were 100.00%. Among 48 simulated clinical samples, the positive coincidence rate reached 97.73%, while maintaining a perfect negative coincidence rate of 100.00%. Moreover, the detection platform holds immense potential for attaining a more comprehensive bioterrorism screening, and its high cost-effectiveness enables the provision of diverse and adaptable diagnostic methods for public health quarantine in underdeveloped countries and regions.