Quantitative Polymerase Chain Reaction Coupled With Sodium Dodecyl Sulfate and Propidium Monoazide for Detection of Viable Streptococcus agalactiae in Milk

Streptococcus agalactiae is an important pathogen causing bovine mastitis. The aim of this study was to develop a simple and specific method for direct detection of S. agalactiae from milk products. Propidium monoazide (PMA) and sodium dodecyl sulfate (SDS) were utilized to eliminate the interference of dead and injured cells in qPCR. Lysozyme (LYZ) was adopted to increase the extraction efficiency of target bacteria DNA in milk matrix. The specific primers were designed based on cfb gene of S. agalactiae for qPCR. The inclusivity and exclusivity of the assay were evaluated using 30 strains. The method was further determined by the detection of S. agalactiae in spiked milk. Results showed significant differences between the SDS–PMA–qPCR, PMA–qPCR and qPCR when a final concentration of 10 mg/ml (R2 = 0.9996, E = 95%) of LYZ was added in DNA extraction. Viable S. agalactiae was effectively detected when SDS and PMA concentrations were 20 μg/ml and 10 μM, respectively, and it was specific and more sensitive than qPCR and PMA–qPCR. Moreover, the SDS–PMA–qPCR assay coupled with LYZ was used to detect viable S. agalactiae in spiked milk, with a limit of detection of 3 × 103 cfu/ml. Therefore, the SDS–PMA–qPCR assay had excellent sensitivity and specificity for detection of viable S. agalactiae in milk

Characterization of RNA editome in primary and metastatic lung adenocarcinomas

RNA editing results in post-transcriptional modification and could potentially contribute to carcinogenesis. However, RNA editing in advanced lung adenocarcinomas has not yet been studied. Based on whole genome and transcriptome sequencing data, we identified 1,071,296 RNA editing events from matched normal, primary and metastatic samples contributed by 24 lung adenocarcinoma patients, with 91.3% A-to-G editing on average, and found significantly more RNA editing sites in tumors than in normal samples. To investigate cancer relevant editing events, we detected 67,851 hyper-editing sites in primary and 50,480 hyper-editing sites in metastatic samples. 46 genes with hyper-editing in coding regions were found to result in amino acid alterations, while hundreds of hyper-editing events in non-coding regions could modulate splicing or gene expression, including genes related to tumor stage or clinic prognosis. Comparing RNA editome of primary and metastatic samples, we also discovered hyper-edited genes that may promote metastasis development. These findings showed a landscape of RNA editing in matched normal, primary and metastatic tissues of lung adenocarcinomas for the first time and provided new insights to understand the molecular characterization of this disease

A targeted next-generation sequencing method for identifying clinically relevant mutation profiles in lung adenocarcinoma

Molecular profiling of lung cancer has become essential for prediction of an individual’s response to targeted therapies. Next-generation sequencing (NGS) is a promising technique for routine diagnostics, but has not been sufficiently evaluated in terms of feasibility, reliability, cost and capacity with routine diagnostic formalin-fixed, paraffin-embedded (FFPE) materials. Here, we report the validation and application of a test based on Ion Proton technology for the rapid characterisation of single nucleotide variations (SNVs), short insertions and deletions (InDels), copy number variations (CNVs), and gene rearrangements in 145 genes with FFPE clinical specimens. The validation study, using 61 previously profiled clinical tumour samples, showed a concordance rate of 100% between results obtained by NGS and conventional test platforms. Analysis of tumour cell lines indicated reliable mutation detection in samples with 5% tumour content. Furthermore, application of the panel to 58 clinical cases, identified at least one actionable mutation in 43 cases, 1.4 times the number of actionable alterations detected by current diagnostic tests. We demonstrated that targeted NGS is a cost-effective and rapid platform to detect multiple mutations simultaneously in various genes with high reproducibility and sensitivity

Preoperative CT-guided ICG injection locating SPNs

Background: Localization of small pulmonary nodules (SPNs) is challenging in minimally invasive pulmonary resection, and it is unknown whether computer tomography (CT) guided by indocyanine green (ICG) can provide accurate localization with minimal complications. Methods: We performed a retrospective study of patients who underwent thoracoscopic resection of pulmonary nodules after CT-guided preoperative localization with ICG from May 2019 to May 2020. Demographics, procedural data, postoperative complications, and pathologic information, were collected, and an analysis of the accuracy and complications after surgery was conducted. Results: In 471 patients, there was a total of 512 peripheral pulmonary nodules that were ≤2 cm in size. The average time for CT-guided percutaneous ICG injection for localization was 18 minutes, and 98.4% (504/512) of the nodules were successfully localized. The average size of the nodules was 9.1 mm, and the average depth from the pleural surface was 8.9 mm. Overall, 5.9% (28/471) of the patients had asymptomatic pneumothorax after localization, but none needed a tube thoracostomy. All the nodules were resected using video-assisted thoracoscopy technique. Conclusions: Preoperative CT-guided transthoracic ICG injection is safe and feasible for localization of small lung nodules for minimally invasive pulmonary resection. This technique should be considered for preoperative CT-guided localization of small lung nodules

Development and application of quadruplex real time quantitative PCR method for differentiation of Muscovy duck parvovirus, Goose parvovirus, Duck circovirus, and Duck adenovirus 3

IntroductionMuscovy duck parvovirus (MDPV), Goose parvovirus (GPV), Duck circovirus, (DuCV) and Duck adenovirus 3 (DAdV-3) are important pathogens that cause high morbidity and mortality in ducks, causing huge economic loss for the duck industry.MethodsThe present study, a quadruplex one-step real time quantitative PCR method for the detection of MDPV, GPV, DuCV, and DAdV-3 was developed. ResultsThe results showed that assay had no cross-reactivity with other poultry pathogens [Duck plague virus (DPV), Duck tembusu virus (DTMUV), H6 avian influenza virus (H6 AIV), New duck reovirus (NDRV), Newcastle disease virus (NDV), H4 avian influenza virus (H4 AIV), Escherichia coli (E. coli), Muscovy duck reovirus (MDRV), Egg drop syndrome virus (EDSV), Pasteurella multocida (P. multocida)]. The sensitivity result showed that the limits of detection for MDPV, GPV, DuCV, and DAdV-3 were 10, 10, 1 and 10 copies/µl, respectively; The coefficients of variation intra- and inter-method was 1-2%; The range of linear (109 to 103 copies/µL) demonstrated the R2 values for MDPV, GPV, DuCV, and DAdV-3 as 0.9975, 0.998, 0.9964, and 0.996, respectively. The quadruplex real time quantitative PCR method efficiency was 90.30%, 101.10%, 90.72%, and 90.57% for MDPV, GPV, DuCV, and DAdV-3, respectively. 396 clinical specimens collected in some duck sausages from June 2022 to July 2023 were simultaneously detected using the established quadruplex real time quantitative PCR method and the reported assays. The detection rates for MDPV, GPV, DuCV, and DAdV-3 were 8.33% (33/396), 17.93% (71/396), 33.58% (133/396), and 29.04% (115/396), respectively. The agreement between these assays was greater than 99.56%.DiscussionThe developed quadruplex real-time quantitative PCR assay can accurately detect these four viruses infecting ducks, providing a rapid, sensitive, specific and accurate technique for clinical testing