Development of Novel Real-Time PCR Assays for Detection and[1]

7500 Real-Time

PCR System Manual

Blackhills Diagnostic Resources S.L.U.

Camino del Pilón 86, casa 7 local - 50011 Zaragoza, Spain

+34 976 094 603 – ***********************

Rev. 01 - 2022/06/10

1 7500 Real-Time PCR System Manual_Rev.01

(+34)976094603·***********************·7500 Real-Time PCR System

Manual

AMPLIFICATION CURVES ANALYSIS

CREATE THE TEMPLATE

Open the software and select New Experiment. To perform further analysis of results with the Genvinset® Report Viewer software, it is important to correctly create the template before the run.

1. Set up the experiment properties and in Experiment type, select Quantitation - Standard Curve.

2. Go to Setup > Plate Setup, a) In the Define Targets and Samples tab, add new targets, name the genes and select the reporter as specified in Figure 2. For multi-reaction kits, add the target genes of all reactions. b) In the Assign targets and Samples tab, select none as passive reference. 3. In the Setup > Run Method tab set the amplification program. 4. To save the setup of the experiment to use it on further occasions, click ok File > Save As Template.

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F1768K Hot Start rTth (DNA) Kit HSTTH-301 250U Store at -20°C Contents [1] Introduction [2] Components [3] Protocol 1. Standard reaction setup 2. Cycling conditions [4] Example [5] Related products CAUTION All reagents in this kit are intended for research purposes. Do not use for diagnosis or clinical purposes. Please observe general laboratory precautions and safety while using this kit. -TaqMan® is a registered trademark of Roche Molecular Systems, Inc. 1 www.toyobo.co.jp/e/bio

JAPAN CHINA TOYOBO CO., LTD. TOYOBO (SHANHAI) BIOTECH CO., LTD. Tel(81)-6-6348-3888 Tel(86)-21-5879-4900 www.toyobo.co.jp/e/bio ******************** [1] Introduction [2] Components Description Hot Start rTth (DNA) Kit is PCR reagent based on Tth DNA Polymerase. Tth DNA Polymerase has higher amplification efficiency than Taq DNA Polymerase, which is a general-purpose enzyme, and enables amplification from low copies of template and amplification from a crude sample containing PCR inhibitors. In addition, Tth DNA Polymerase has a 5 '- 3' exonuclease activity, so it can be used for real-time PCR using probe assays such as TaqMan® assay. This enzyme contains neutralizing antibodies, thus allowing for Hot start PCR. Features -Excellent DNA Amplification Efficiency The reaction composition is optimized based on Tth DNA Polymerase, enabling efficient PCR even from low copies of template. - Tolerant of PCR Inhibitors This kit is effective for amplification from crude samples (e.g., biological samples, foodstuffs, soil extract, etc.). In the case of amplification from whole blood, sufficient amplification can be achieved by adding it directly to the reaction solution without purification of nucleic acid. - Utilization of dUTP This kit contains dUTP instead of dTTP in 2x Buffer for rTth/ TTx (DNA). Therefore, the rate of false-positive detection can be reduced by adding uracil-N-glycosylase (UNG). *UNG is not supplied with this kit. This kit includes the following components for 250 reactions, 20 μL total reaction volume. All reagents should be stored at -20°C. 2x Buffer for rTth/ TTx (DNA) 1.25 mLx 2 Hot Start rTth DNA Polymerase (4U/ μL) 62.5 μL Note: -2× Reaction Buffer contains essential components for the reaction (buffer, salts, Mg2+, dATP, dCTP, dGTP, and dUTP, etc.). Add template DNA, primers, and attached Hot Start rTth DNA Polymerase, and adjust to 1x concentration with sterile water etc. -DNA Polymerase is a mixture of rTth DNA polymerase and hot start antibodies. Its concentration is 4U/ μL. -This kit doesn’t contain a passive reference dye (ROX). When using a passive reference dye to compensate fluorescence intensity and dispensing error between wells, please use the separately sold 50x ROX reference dye (Code No. ROX-101).

23320212362 NGS检出脑脊液样本中布尼亚病毒1例报道及文献复习石玉如1， 冯　刚2， 岳　莉1， 谷　妍1， 刘　杨1， 戚应杰1（1.安徽省立医院感染病院，安徽 合肥 230022；2.安徽省金寨县人民医院，安徽 金寨 237300）关键词：下一代测序；布尼亚病毒；感染；脑脊液

基金项目：中国公共卫生联盟科研基金资助项目（GWLM202012）。作者简介：石玉如，女，1984年生，硕士，主管技师，主要从事临床感染性疾病的免疫与分子诊断研究。通信作者：戚应杰， E-mail：。文章编号：1673-8640（2021）02-0233-04 中图分类号：R446.1 文献标志码：B DOI：10.3969/j.issn.1673-8640.2021.02.025

发热伴血小板减少综合征（severe fever with thrombocytopenia syndrome，SFTS）是由布尼亚病毒[又称发热伴血小板减少综合症病毒（Severe fever with thrombocytopenia virus，SFTSV）]导致的以发热伴血小板减少为主要临床特征的感染性疾病。在流行区域该病毒感染率为1%～3%，病死率为6%～30%，平均病死率约为10%[1]。该病毒可通过蜱虫、饲养或野生动物、人与人血液或分泌物传播[2]，好发于疫区农民，患者大多住在丘陵或森林地带。该病主要流行于5～7月，5月为发病高峰期。继中国后，韩国、日本、美国等国也有相关报道，SFTSV感染的防治已成为全球性公共卫生问题，不可忽视[3]。本研究采用高通量下一代测序（next-generation sequencing，NGS）技术对1例疑似SFTSV感染重症患者的脑脊液样本进行测序，明确病毒病原体，并结合临床特征及相关实验室检查结果，提高对SFTSV感染重症的认识，同时探讨NGS在脑脊液病原体感染诊断中的价值。1 病例资料患者，女，50岁，汉族，安徽安庆地区居民。2018年4月4日采茶时被虫咬伤左髋部，未经特殊处理，随后出现腹泻、头昏、乏力、高热和畏寒症状，最高体温39 ℃。4月6日至当地镇医院检查发现血小板偏低，转至县医院消化科住院，检查发现白细胞和血小板进一步降低，血压降低，4月7日转至市级医院检查发现血小板36×109/L，白细胞2.02×109/L，D-二聚体> 20 μg/mL，丙氨酸氨基转移酶1 034 IU/L，肌钙蛋白24 pg/mL，予以抗感染、保肝、补液扩容等对症处理后，仍有胸闷、气促等症状，4月10日以“发热伴血小板减少”收入安徽省立医院。入院时昏迷状态，体温36.8 ℃，全身多处皮下瘀斑，呼吸急促，双肺少量湿啰音；腹平软，肠鸣音存在。4月11日予以腰椎穿刺进行脑脊液NGS、细菌培养及常规检查，并立即行升压、保护脏器及抗感染等对症治疗，严密监测生命体征，患者仍出现抽搐、意识障碍现象；4月12日上午患者再次出现抽搐，伴有血氧饱和度下降，对症治疗后，抽搐好转；4月13日心电监护提示血压低，心率约110次/min，行气管内插管呼吸机辅助通气过程中，血乳酸明显升高，氧分压低，脑脊液压力极低，积极予以维持血压、抗休克、气管插管呼吸机辅助通气、深静脉置管、扩容、补液等对症支持治疗，白细胞和血小板上升，但仍需大剂量升压药物维持，仍存在意识障碍，偶见自主睁眼。不同时间点的实验室检查结果见表1。2 实验室检查2.1 脑脊液NGS结果 检出SFTSV HB29，检出序列数最多为 1 321。检测到SFTSV HB29唯一比对序列数为112条，覆盖度达97.82%，见图1。2.2 SFTSV核酸检测结果SFTSV核酸检测试剂盒购自上海之江生物公司，以自带标准品（S0=0拷贝/mL、S1=1.0× 104拷贝/mL、S2=1.0×105拷贝/mL、S3=1.0× 106拷贝/mL、S4=1.0×107拷贝/mL）绘制标准曲线，患者样本检测结果为5.2×108拷贝/mL。 见图2。23420212362 表1　患者不同时间相关实验室检查结果时间白细胞计数/ ×109/L血小板计数/ ×109/L丙氨酸氨基转移酶/（IU/L）天门冬氨酸氨基转移酶/（IU/L）降钙素原/（ng/mL）氨基末端B型钠尿肽原/（pg/mL）乳酸脱氢酶/（U/L）4月10日1.993826713242.1112920944月11日3.22302.63537230474月12日2.192999854月13日7.5534月16日

Abstract:

The rapid spread of viral infections has posed significant challenges to

global public health. In this study, we evaluated the efficacy of a

novel antiviral drug, AV-123, against a panel of viral strains in vitro.

The drug was tested on various cell lines, and its inhibitory effects

were measured using the viral plaque assay and quantitative real-time

PCR. The results demonstrated that AV-123 exhibited potent antiviral

activity against the tested viruses, with minimal cytotoxicity. This

study provides a foundation for further investigation of AV-123 as a

potential therapeutic agent against viral infections.

Introduction:

Viral infections are a major cause of morbidity and mortality worldwide.

The emergence of drug-resistant strains has further complicated the

management of these infections. Antiviral drugs are essential in