Real-time Fluorescent Reverse Transcription Polymerase Research Articles

Development of an Integrated Disposable Device for SARSCoV-2 Nucleic Acid Extraction and Detection

ObjectiveTo develop a highly sensitive and rapid nucleic acid detection method for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). MethodsWe designed, developed, and manufactured an integrated disposable device for SARS-CoV-2 nucleic acid extraction and detection. The precision of the liquid transfer and temperature control was tested. A comparison between our device and a commercial kit for SARS-Cov-2 nucleic acid extraction was performed using real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR). The entire process, from SARS-CoV-2 nucleic acid extraction to amplification, was evaluated. ResultsThe precision of the syringe transfer volume was 19.2 ± 1.9 μL (set value was 20), 32.2 ± 1.6 (set value was 30), and 57.2 ± 3.5 (set value was 60). Temperature control in the amplification tube was measured at 60.0 ± 0.0 °C (set value was 60) and 95.1 ± 0.2 °C (set value was 95) respectively. SARS-Cov- 2 nucleic acid extraction yield through the device was 7.10 × 106 copies/mL, while a commercial kit yielded 2.98 × 106 copies/mL. The mean time to complete the entire assay, from SARS-CoV-2 nucleic acid extraction to amplification detection, was 36 min and 45 s. The detection limit for SARS-CoV-2 nucleic acid was 250 copies/mL. ConclusionThe integrated disposable devices may be used for SARS-CoV-2 Point-of-Care test (POCT).

Quantitative CT for detecting COVID\u201119 pneumonia in suspected cases

BackgroundCorona Virus Disease 2019 (COVID-19) is currently a worldwide pandemic and has a huge impact on public health and socio-economic development. The purpose of this study is to explore the diagnostic value of the quantitative computed tomography (CT) method by using different threshold segmentation techniques to distinguish between patients with or without COVID-19 pneumonia.MethodsA total of 47 patients with suspected COVID-19 were retrospectively analyzed, including nine patients with positive real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR) test (confirmed case group) and 38 patients with negative RT-PCR test (excluded case group). An improved 3D convolutional neural network (VB-Net) was used to automatically extract lung lesions. Eight different threshold segmentation methods were used to define the ground glass opacity (GGO) and consolidation. The receiver operating characteristic (ROC) curves were used to compare the performance of various parameters with different thresholds for diagnosing COVID-19 pneumonia.ResultsThe volume of GGO (VOGGO) and GGO percentage in the whole lung (GGOPITWL) were the most effective values for diagnosing COVID-19 at a threshold of − 300 HU, with areas under the curve (AUCs) of 0.769 and 0.769, sensitivity of 66.67 and 66.67%, specificity of 94.74 and 86.84%. Compared with VOGGO or GGOPITWL at a threshold of − 300 Hounsfield units (HU), the consolidation percentage in the whole lung (CPITWL) with thresholds at − 400 HU, − 350 HU, and − 250 HU were statistically different. There were statistical differences in the infection volume and percentage of the whole lung, right lung, and lobes between the two groups. VOGGO, GGOPITWL, and volume of consolidation (VOC) were also statistically different at the threshold of − 300 HU.ConclusionsQuantitative CT provides an image quantification method for the auxiliary diagnosis of COVID-19 and is expected to assist in confirming patients with COVID-19 pneumonia in suspected cases.

Epidemiological investigation of a COVID-19 family cluster outbreak transmitted by a 3-month-old infant

ObjectiveTo investigate the clinical characteristics, epidemiological characteristics, and transmissibility of coronavirus disease 2019 (COVID-19) in a family cluster outbreak transmitted by a 3-month-old confirmed positive infant.MethodsField-based epidemiological methods were used to investigate cases and their close contacts. Real-time fluorescent reverse transcription polymerase chain reaction (RT-PCR) was used to detect Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) for all collected specimens. Serum SARS-CoV-2 IgM and IgG antibodies were detected by Chemiluminescence and Gold immnnochromatography (GICA).ResultsThe outbreak was a family cluster with an attack rate of 80% (4/5). The first case in this family was a 3-month-old infant. The transmission chain was confirmed from infant to adults (her father, mother and grandmother). Fecal tests for SARS-CoV-2 RNA remained positive for 37 days after the infant was discharged. The infant’s grandmother was confirmed to be positive 2 days after the infant was discharged from hospital. Patients A (3-month-old female), B (patient A’s father), C (patient A’s grandmother), and D (patient A’s mother) had positive serum IgG and negative IgM, but patients A’s grandfather serum IgG and IgM were negative.ConclusionSARS-CoV-2 has strong transmissibility within family settings and presence of viral RNA in stool raises concern for possible fecal–oral transmission. Hospital follow-up and close contact tracing are necessary for those diagnosed with COVID-19.

Results analysis on SARS-CoV-2 nucleic acid detection based on different target gene detection

Objective To compare the results of three SARS-CoV-2 nucleic acid detection reagents (reagent A, B and based on different target gene detection, and we analyze the consistency by preliminary evaluation of the application of each reagent. Methods Totally 43 samples of pharyngeal swabs were collected from our hospital and the designated treatment hospital, and detected by the real-time fluorescencereverse transcription-polymerase chain reaction (RT-PCR) in three SARS- CoV-2 nucleic acid detection reagent (reagent A, B and C) at the same time. Clinical diagnosis was divided into COVID-19 group ( n =31) and control group ( n =12). Fisher exact probability test and Kendall coordination coefficient W test were used to test the difference of positive rate and consistency of test results. Results The positive detection rate of reagent B was the highest at 90.32%, and there was no reexamination result.The positive detection rate of reagent A and reagent C were 77.42% and 74.19%, and more reexamination results were needed for reagent C. In the control group, reagent A and C did not test positive, but reagent B showed one positive result. The difference of positive detection rate among reagents was not statistically significant ( P >0.05), and the difference in consistency was statistically significant, with progressive significance Sig=0.00 and Kendall’s W coefficient value being 0.852, indicating that the consistency of test results of the three reagents was very high. Conclusion The three reagents showed good consistency in the nucleic acid test results for COVID-19. Reagent B has advantages in sensitivity, and reagent A and C are stable in specificity.Reagent B is most suitable for strict screening during an outbreak, but false positives should be noted. Reagent A and reagent C need to improve the quality of test samples for false negative and reexamination results, and the result interpretation method of reagent C is not good in the processing of large quantities of positive sample data. 摘要： 目的 比较三种不同靶基因检测 2019 新型冠状病毒(SARS-CoV-2)的检测结果，分析其一致性，初步评估各 试剂的应用情况。 方法 收集 43 份本院患者和定点治疗医院送来的咽拭子标本，根据临床诊断分为新型冠状病毒肺 炎(COVID-19)组 31 份和对照组 12 份，用三种 SARS-CoV-2 核酸检测试剂同时进行实时荧光逆转录聚合酶链反应(re⁃ verse transcription-polymerase chain reaction, RT-PCR)检测，检测结果的阳性率差异和一致性分别用 Fisher 精确概率检 验和 Kendall’s 协调系数 W 检验。 结果 在 COVID-19 组中试剂 B 的阳性检出率最高为 90.32%，且不存在需复检结果； 试剂 A 和试剂 C 的阳性检出率分别为 77.42% 和 74.19%，且试剂 C 出现的需复检结果较多；在对照组中试剂 A 和试剂 C 未检测出阳性，但试剂 B 出现一例阳性结果。COVID-19 组中各试剂的阳性检出率差异不具有统计学意义( P >0.05)；一 致性方面差异具有统计学意义，渐进显著性 Sig=0.00，Kendall’sW 系数值为 0.852，说明三种试剂检测结果的一致性程度 很高。 结论 三种试剂对 COVID-19 核酸检测的结果一致性很好，灵敏度方面试剂 B 较有优势，特异性方面试剂 A 和 试剂 C 较稳定。试剂 B 最适合疫情期间的严格排查，但应注意假阳性结果的出现；试剂 A 和试剂 C 针对假阴性和需复 检结果需要提高检测样本的质量，且试剂 C 的结果判读方式在处理大批量阳性标本数据时体验欠佳。

Efficacy of chloroquine versus lopinavir/ritonavir in mild/general COVID-19 infection: a prospective, open-label, multicenter, randomized controlled clinical study

BackgroundThe outbreak of COVID-19 (caused by SARS-Cov-2) is very serious, and no effective antiviral treatment has yet been confirmed. The adage “old drug, new trick” in this context may suggest the important therapeutic potential of existing drugs. We found that the lopinavir/ritonavir treatment recommended in the fifth edition of the Treatment Plan of China can only help to improve a minority of throat-swab nucleic-acid results (3/15) in hospitals. Our previous use of chloroquine to treat patients with COVID-19 infection showed an improvement in more throat-swab nucleic-acid results (5/10) than the use of lopinavir/ritonavir.Methods/designThis is a prospective, open-label, randomized controlled, multicenter clinical study. The study consists of three phases: a screening period, a treatment period of no more than 10 days, and a follow-up period for each participant. Participants with COVID-19 infection who are eligible for selection for the study will be randomly allocated to the trial group or the control group. The control group will be given lopinavir/ritonavir treatment for no more than 10 days. The trial group will be given chloroquine phosphate treatment for no more than 10 days. The primary outcome is the clinical recovery time at no more than 28 days after the completion of therapy and follow-up. The secondary outcomes include the rate of treatment success after the completion of therapy and follow-up, the time of treatment success after no more than 28 days, the rate of serious adverse events during the completion of therapy and follow-up, and the time to return to normal temperature (calculated from the onset of illness) during the completion of therapy and follow-up. Comparisons will be performed using two-sided tests with a statistical significance level of 5%.DiscussionThis experiment should reveal the efficacy and safety of using chloroquine versus lopinavir/ritonavir for patients with mild/general COVID-19 infection. If the new treatment including chloroquine shows a higher rate of throat-swab SARS-CoV-2 real-time fluorescent reverse transcription polymerase chain reaction (RT-PCR) negativity and is safe, it could be tested as a future COVID-19 treatment.Trial registrationChinese Clinical Trial Registry, ID: ChiCTR2000029741. Registered on 11 February 2020.

Pathogen spectrum in enteroviral infections among children in Beijing from 2010 to 2016

Objective: To understand the epidemiological and etiological characteristics of enterovirus (EV)-associated diseases among children in Beijing from 2010 to 2016. Methods: This was a repeated cross-sectional study. The throat swabs were collected from children with probable EV-associated diseases at the Children' s Hospital Affiliated to Capital Institute of Pediatrics from 2010 to 2016. The samples were sent for pan-EV, enterovirus 71 (EV-A71) and coxsackievirus A16 (CV-A16) detection by real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR) . The viral types of non-EV-A71 and non-CV-A16 EV-positive samples were identified using modified RT-PCR and sequencing with CV-A6, EV-A/B group and 5 'UTR universal primers. The constituent ratios of the prevalence of different EV types in different age and gender groups were compared. Results: Of the 2 703 throat swabs, 1 992 (73.7%) samples were positive for EV, including EV-A71 (19.1%, 516/2 703), CV-A16 (24.3%, 658/2 703), CV-A6 (22.2%, 600/2 703), CV-A10 (4.5%, 122/2 703) and other types of EV (3.5%, 95/2 703). There was 1 case of EV-A71 and CV-A16 co-infection. The positive detection rate of EV-A group (excluding EV-A71, CV-A16, CV-A6 and CV-A10) increased from 11.3% (7/62) to 95.2% (59/62) after using the modified VP1-specific primers and PCR amplification conditions. During the period between 2010 and 2012, CV-A16 and EV-A71 predominated in EV-positive samples. However, CV-A6 accounted for 60.7% (68/112) in 2013, much higher than CV-A16 (23.2%, 26/112) and EV-A71 (12.5%, 14/112). In 2014, EVs were mainly of CV-A16 and EV-A71, but CV-A6 was the predominant type in 2015 (68.2%, 232/340) and in 2016 (38.6%, 151/391). The epidemic season of EVs was mostly from April to August, but CV-A6 showed a small epidemic peak from October to November. The male-to-female ratio of EV-positive patients was 1.50∶1, and EV-associated diseases mostly occurred in children under 5 years of age. Younger children were more susceptible to CV-A6 than to EV-A71 and CV-A16. Conclusions: From 2010 to 2016, there was a significant change in the spectrum of EVs in children with EV-associated diseases in Beijing. Since 2013, non-EV-A71 and non-CV-A16 increased, and CV-A6 gradually became one of the major pathogens of EV-associated diseases. The modified PCR primers and amplification conditions can effectively improve the reliability of test results.

Effects of Angiotensin Inhibitor Valsartan on the Expression of the Angiotensin II 1 Receptor, Matrix Metalloproteinases -2 and -9 in Human Bladder Cancer Cell Lines.

In order to investigate how valsartan-the angiotensin II 1 receptor (AT1R) antagonist-affects the expressions of AT1R antigen, matrix metalloproteinases (MMPs) -2 and -9 in carcinoma of urinary bladder (CUB) cell lines with different invasive abilities. Three cell lines, EJ-M3, EJ, and BIU-87, with different invasive abilities were cultured and treated with valsartan. Cell proliferation states were determined by the methyl thiazolyl tetrazolium (MTT) method. The expressions at protein level and gene level were determined by Western blot and real-time fluorescence reverse transcription polymerase chain reaction (RT-PCR), respectively. The invasive abilities and migratory abilities of the three cell lines were determined by Transwell in vitro cell invasion assay and wound healing assay, respectively. MTT results show that valsartan can inhibit the proliferation of CUB cells, and the inhibition effect is enhanced with the increase of concentration. AngII promotes the MMP2 and MMP9 expressions (both protein and gene levels) in CUB cells through AT1R, but their expressions can be effectively inhibited by valsartan, the AngII inhibitor. AngII inhibitor may become a novel drug that can inhibit CUB metastasis and prolong the survival of CUB patients.

Beet necrotic yellow vein virus (benyvirus)

<i>Beet necrotic yellow vein virus</i> (<i>benyvirus</i>)