Establishment and performance analysis of a new multiplex detection method for influenza an and B virus antigen.

BACKGROUND: Influenza A and B virus detection is pivotal in epidemiological surveillance and disease management. Rapid and accurate diagnostic techniques are crucial for timely clinical intervention and outbreak prevention. Quantum dot-encoded microspheres have been widely used in immunodetection. The integration of quantum dot-encoded microspheres with flow cytometry is a well-established technique that enables rapid analysis. Thus, establishing a multiplex detection method for influenza A and B virus antigens based on flow cytometry quantum dot microspheres will help in disease diagnosis. AIM: To establish a codetection method of influenza A and B virus antigens based on flow cytometry quantum dot-encoded microsphere technology, which forms the foundation for the assays of multiple respiratory virus biomarkers. METHODS: Different quantum dot-encoded microspheres were used to couple the monoclonal antibodies against influenza A and B. The known influenza A and B antigens were detected both separately and simultaneously on a flow cytometer, and the detection conditions were optimized to establish the influenza A and B antigen codetection method, which was utilized for their detection in clinical samples. The results were compared with the fluorescence quantitative polymerase chain reaction (PCR) method to validate the clinical performance of this method. RESULTS: The limits of detection of this method were 26.1 and 10.7 pg/mL for influenza A and B antigens, respectively, which both ranged from 15.6 to 250000 pg/mL. In the clinical sample evaluation, the proposed method well correlated with the fluorescent quantitative PCR method, with positive, negative, and overall compliance rates of 57.4%, 100%, and 71.6%, respectively. CONCLUSION: A multiplex assay for quantitative detection of influenza A and B virus antigens has been established, which is characterized by high sensitivity, good specificity, and a wide detection range and is promising for clinical applications.

Non-invasive prenatal screening for foetal trisomy: An assessment of reliability and reporting.

OBJECTIVE: Non-invasive prenatal screening (NIPS) has been introduced as a routine screening technique for aneuploidies in the clinic. To evaluate its reliability and reporting standardization, the National Center for Clinical Laboratories launched an external quality assessment (EQA) program based on highly simulated samples. METHODS: Maternal and child paired cell lines were digested by enzymes to obtain DNA fragments for the analysis panel, which were composed of 5% T21, 5% T18, 10% T13, 10% euploid, and 20% T18 samples. The samples were validated and distributed to laboratories along with scenarios and questionnaires for analysis. RESULTS: Out of 350 participating laboratories, 98.6% correctly identified all samples. The concurrence rates of laboratories for the 5% T21, 5% T18, 10% T13, 10% euploid, and 20% T18 samples were 98.9%, 99.7%, 99.7%, 100%, and 100%, respectively. Enrichment increased the foetal fraction (FF) values by 2 âˆ¼ 3-fold, but the z scores generated by the enrichment group fluctuated greatly. Other FF estimation techniques, such as the size-based and FF-QuantSC methods, generated slightly different FF values from the chr Y-based method. Furthermore, some laboratories omitted the suggestions of results in reports. CONCLUSIONS: The participating laboratories provided highly reliable results for samples with relatively higher FF values. However, the absence of performance validation, laboratory errors, and low FF values were potential reasons for false-negative results. In addition, enrichment operations should be validated and normalized to guarantee NIPS reproducibility, plus further efforts are required to standardize the NIPS reports.

CircMTO1 suppresses hepatocellular carcinoma progression via the miR-541-5p/ZIC1 axis by regulating Wnt/ß-catenin signaling pathway and epithelial-to-mesenchymal transition.

CircRNA mitochondrial tRNA translation optimization 1 (circMTO1) functions as a tumor suppressor usually and is related to the progression of many tumors, including hepatocellular carcinoma (HCC). CircMTO1 is downregulated in HCC as compared to adjacent nontumor tissue, which may suppress the HCC progression by certain signal pathways. However, the underlying signal pathway remains largely unknown. The interactions between circMTO1 and miR-541-5p were predicted through bioinformatics analysis and verified using pull-down and dual-luciferase reporter assays. CCK-8, transwell, and apoptosis assays were performed to determine the effect of miR-541-5p on HCC progression. Using bioinformatic analysis, dual-luciferase reporter assay, RT-qPCR, and western blot, ZIC1 was found to be the downstream target gene of miR-541-5p. The regulatory mechanisms of circMTO1, miR-541-5p, and ZIC1 were investigated using in vitro and in vivo rescue experiments. The results depicted that silencing circMTO1 or upregulating miR-541-5p expression facilitated HCC cell proliferation, migration, and invasion and inhibited apoptosis. CircMTO1 silencing upregulated the expression of downstream ZIC1 regulators of the Wnt/ß-catenin pathway markers, ß-catenin, cyclin D1, c-myc, and the mesenchymal markers N-cadherin, Vimentin, and MMP2, while the epithelial marker E-cadherin was downregulated. MiR-541-5p knockdown had the opposite effect and reversed the effect of circMTO1 silencing on the regulation of downstream ZIC1 regulators. Intratumoral injection of miR-541-5p inhibitor suppressed tumor growth and reversed the effect of circMTO1 silencing on the promotion of tumor growth in HCC. These findings indicated that circMTO1 suppressed HCC progression via the circMTO1/ miR-541-5p/ZIC1 axis by regulating Wnt/ß-catenin signaling and epithelial-to-mesenchymal transition, making it a novel therapeutic target.

External quality assessment of molecular testing of 9 viral encephalitis-related viruses in China.

BACKGROUND: Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), Venezuelan equine encephalitis virus (VEEV), Hendra virus (HeV), Nipah virus (NiV), Yellow fever virus (YFV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV) and Tick-borne encephalitis virus (TBEV) have been detected in travelers returning to China and potentially pose a serious threat to public health. Real-time reverse transcription polymerase chain reaction (rRT-PCR) plays an important role in the detection of these viruses. Although these viruses are not mainly prevalent in China, occasionally imported cases have been reported with the increase in population mobility and entry-exit activities. Therefore, it is necessary to monitor the ability of major domestic laboratories to detect and identify exotic arbovirus infections in travelers. METHODS: An external quality assessment program for the molecular detection of EEEV, VEEV, WEEV, SLEV, WNV, YFV, TBEV, HeV and NiV was organized. The assessment panel included 26 negative and positive samples with different concentrations of virus-like particles and distributed to 31 laboratories to evaluate the accuracy of virus detection. RESULTS: At the laboratory level, 87.5% (7/8, EEEV), 85.7% (12/14, WEEV), 100% (13/13, VEEV), 87.5% (7/8, HeV), 76.5% (13/17, NiV), 92.6% (25/27, YFV), 81.3% (13/16, WNV), 100% (5/5, SLEV) and 75.0% (6/8, TBEV) of the participants were considered "competent". Of all the results, the false-positive and false-negative rates were 0.3% and 0.7%, respectively. The sensitivity of most detection assays (15/17, 88.2%) was more than 90%. In addition, we observed significantly different cycle threshold values when using primer-probe sets in different target regions to detect EEEV and SLEV. CONCLUSIONS: Most laboratories have reliable virus detection capabilities. However, laboratory testing capabilities need to be improved to avoid cross-contamination and to better manage undetected false-negative samples.

Comparison of analytical sensitivity of SARS-CoV-2 molecular detection kits.

OBJECTIVES: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has had a significant impact on global public health systems, making nucleic acid detection an important tool in epidemic prevention and control. Detection kits based on real-time reverse transcriptase PCR (rRT-PCR) have been used widely in clinics, but their analytical sensitivity (limit of detection, LOD) remains controversial. Moreover, there is limited research evaluating the analytical sensitivity of other molecular detection kits. METHODS: In this study, armored ribonucleic acid reference materials developed in-house were used to evaluate the analytical sensitivity of SARS-CoV-2 detection kits approved by the National Medical Products Administration. These were based on rRT-PCR and other molecular detection assays. RESULTS: The percentage retesting required with rRT-PCR kits was as follows: 0%, 7.69%, 15.38%, and 23.08% for samples with concentrations ranging from 50 000 to 781 copies/ml. In total, 93% of rRT-PCR kits had a LOD <1000 copies/ml. Only one kit had an LOD >1000 copies/ml. The LOD of other molecular detection kits ranged from 68 to 2264 copies/ml. CONCLUSIONS: The study findings can help pharmaceutical companies optimize and improve detection kits, guide laboratories in selecting kits, and assist medical workers in their daily work.

Development of novel quality control material based on CRISPR/Cas9 editing and xenografts for MLH1 protein deficiency testing.

BACKGROUND: Mismatch repair deficiency (dMMR) status induced by MLH1 protein deficiency plays a pivotal role in therapeutic decision-making for cancer patients. Appropriate quality control (QC) materials are necessary for monitoring the accuracy of MLH1 protein deficiency assays used in clinical laboratories. METHODS: CRISPR/Cas9 technology was used to edit the MLH1 gene of GM12878Cas9 cells to establish MLH1 protein-deficient cell lines. The positive cell lines were screened and validated by Sanger sequencing, Western blot (WB), and next-generation sequencing (NGS) and were then used to prepare formalin-fixed, paraffin-embedded (FFPE) samples through xenografting. These FFPE samples were tested by hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) for suitability as novel QC materials for MLH1 protein deficiency testing. RESULTS: We successfully cultured 358 monoclonal cells, with a survival rate of 37.3% (358/960) of the sorted monoclonal cells. Through Sanger sequencing, cell lines with MLH1 gene mutation were identified. Subsequently, two cell lines with MLH1 protein deficiency were identified by WB and named as GM12878Cas9_6 and GM12878Cas9_10. The NGS results further confirmed that the MLH1 gene mutation in these two cell lines would cause the formation of stop codons and terminate the expression of the MLH1 protein. The H&E staining and IHC results also verified the deficiency of the MLH1 protein, and FFPE samples from xenografts proved their similarity and consistency with clinical samples. CONCLUSIONS: We successfully established MLH1 protein-deficient cell lines. Followed by xenografting, we developed novel FFPE QC materials with homogenous, sustainable, and typical histological structures advantages that are suitable for the standardization of clinical IHC methods.

Reliable assessment of BRCA1 and BRCA2 germline variants by next-generation sequencing: a multicenter study.

BACKGROUND: BRCA1/2 gene mutation testing, based on next-generation sequencing (NGS), has been gradually applied in the clinic to serve as preventive early screening for predisposed individuals or to provide treatment options for patients with hereditary breast or ovarian cancers. Here, we evaluated the accuracy of NGS-based mutation detection in BRCA1/2 and the consistency in variant interpretation among clinical laboratories to find the possible reasons underlying inaccurate results and discrepant variant interpretation. METHODS: Laboratories were asked to use their routine procedures to detect six mimetic DNA samples with different BRCA1/2 germline variants. The results of variant detection were required to be submitted via a web-based evaluation system and were automatically scored, according to predefined criteria. The variant interpretation report, including the detailed clinical evidence, was summarized and analyzed for reasons underlying inconsistent results. RESULTS: Overall, only 55.2% (16/29) of laboratories, whose detection score was higher than 90 points, was found to be an acceptable detection capability level. 82.9% (29/35) of the errors were genotype errors. The variant classification results were generally consistent, and 77.8% (7/9) of the variants were given the consistent classification answer. Only two single nucleotide variants (SNVs) had a discrepant classification opinion across laboratories. CONCLUSIONS: The BRCA1/2 variant detection performance should be further improved, especially in reporting the correct genome coordinates. Inconsistent variant classification may be a result of the different clinical pieces of evidence collected by the laboratories. However, discordant clinical evidence also appeared within the same classification results. Therefore, our study provided clear clinical evidence assessment strategies for BRCA1/2 variants, which was aimed at obtaining a consistent variant classification strategy for providing accurate clinical reports to the clinicians.

External Quality Assessment for Molecular Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Clinical Laboratories.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a huge threat to public health. Viral nucleic acid testing is the diagnostic gold standard and can play an important role in the prevention and control of this infection. In this study, bacteriophage MS2 virus-like particles encapsulating specific RNA sequences of SARS-CoV-2 and other coronaviruses were prepared by genetic engineering. The assessment panel, consisting of four positive samples with concentrations of 2.8, 3.5, 4.2, and 4.9 log10 copies/mL and five negative samples with other human coronaviruses, was prepared and distributed to evaluate the accuracy of routine viral RNA detection. Results of 931 panels from 844 laboratories were collected. The overall percentage agreement, positive percentage agreement (PPA), and negative percentage agreement, defined as the percentage of agreement between the correct results and total results submitted for all, positive, and negative samples were 96.8% (8109/8379), 93.9% (3497/3724), and 99.1% (4612/4655), respectively. For samples with concentrations of 4.9 and 4.2 log10 copies/mL, the PPAs were >95%. However, for 3.5 and 2.8 log10 copies/mL, the PPAs were 94.6% (881/931) and 84.9% (790/931), respectively. For all negative samples, the negative percentage agreement values were >95%. Thus, most laboratories can reliably detect SARS-CoV-2. However, further improvement and optimization are required to ensure the accuracy of detection in panel members with lower concentrations of viral RNA.

Quality evaluation of molecular diagnostic tests for astrovirus, sapovirus and poliovirus: A multicenter study.

BACKGROUND: Astrovirus (AstV), Sapovirus (SaV) and Poliovirus (PV) are important pathogens that cause infections in children under five years of age. It is a very important task to systematically monitor and evaluate the diagnostic performance of these viruses in clinical laboratories. METHODS: In our study, we performed a multicenter evaluation study among 21 laboratories across China using simulated stool samples spiked with self-designed AstV, SaV and PV pseudoviral particles. RESULTS: The testing capability of 80.0% (16/20, AstV), 52.6% (10/19, SaV), and 25.0% (2/8, PV) of the participating laboratories were found to be "competent" in reporting correct results for all samples. The main type of errors were false negatives. None of the laboratories identified the subtypes of AstV and SaV, and six laboratories specifically identified the subtypes of PV. Lacking of well-trained personnel and adequate funding were the main challenges. From the questionnaire results, 55.6% laboratories (10/18) believe that training personnel could improve the laboratory testing performance. CONCLUSIONS: The laboratories showed a competent diagnostic performance for AstV, but inferior diagnostic performances for SaV and PV. Sensitivity of detection and the ability for virus typing should be improved clinically. Professional and standardized personnel training is urgently needed to further improve laboratory performance.

Molecular and Serological Assays for SARS-CoV-2: Insights from Genome and Clinical Characteristics.

BACKGROUND: The ongoing outbreak of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed a challenge for worldwide public health. A reliable laboratory assay is essential both to confirm suspected patients and to exclude patients infected with other respiratory viruses, thereby facilitating the control of global outbreak scenarios. CONTENT: In this review, we focus on the genomic, transmission, and clinical characteristics of SARS-CoV-2, and comprehensively summarize the principles and related details of assays for SARS-CoV-2. We also explore the quality assurance measures for these assays. SUMMARY: SARS-CoV-2 has some unique gene sequences and specific transmission and clinical features that can inform the conduct of molecular and serological assays in many aspects, including the design of primers, the selection of specimens, and testing strategies at different disease stages. Appropriate quality assurance measures for molecular and serological assays are needed to maintain testing proficiency. Because serological assays have the potential to identify later stages of the infection and to confirm highly suspected cases with negative molecular assay results, a combination of these two assays is needed to achieve a reliable capacity to detect SARS-CoV-2.