Journal of virological methods最新文献_第10页

Plate centrifugation enhances the efficiency of polyethylenimine-based transfection and lentiviral infection 平板离心提高了聚乙烯亚胺转染和慢病毒感染的效率。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-30 DOI: 10.1016/j.jviromet.2024.115039

Shaozhe Yang , Qingwei Zhang , Yuan Zhuang , Junfeng Li , Xiuhong Fu

{"title":"Plate centrifugation enhances the efficiency of polyethylenimine-based transfection and lentiviral infection","authors":"Shaozhe Yang , Qingwei Zhang , Yuan Zhuang , Junfeng Li , Xiuhong Fu","doi":"10.1016/j.jviromet.2024.115039","DOIUrl":"10.1016/j.jviromet.2024.115039","url":null,"abstract":"<div><h3>Purpose</h3><div>To propose an efficient, reproducible, and consistent transgenic technology based on plate centrifugation, which is particularly useful for polyethylenimine (PEI) transfection and lentiviral infection.</div></div><div><h3>Methods</h3><div>We optimized multiple factors that could contribute to transfection efficiency, such as the dosage of the PEI or DNA, the working solution buffer used for diluting the PEI or DNA, the incubation time for the PEI/DNA complexes, and the transfection time.</div></div><div><h3>Results</h3><div>Plate centrifugation led to a 5.46-fold increase in the transfection efficiency of PEI-based transfection while maintaining the cell survival rate. Moreover, the average copy number of viral genes in each genome increased 4.96-fold with plate centrifugation. Plate centrifugation alters the spatial arrangement of the PEI/DNA complexes or lentiviruses, increasing the chances of these complexes or viruses coming into contact with target cells, ultimately resulting in improved transfection or infection efficiency.</div></div><div><h3>Conclusions</h3><div>We present a protocol based on plate centrifugation for transfection or lentiviral infection that is suitable for genetic modification of primary cells or stem cells.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115039"},"PeriodicalIF":2.2,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A method for producing protease pS273R of the African swine fever virus 一种生产非洲猪瘟病毒蛋白酶 pS273R 的方法

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-24 DOI: 10.1016/j.jviromet.2024.115037

Danil S. Kalinin , Sergey G. Mayorov , Marina Yu. Zemskova , Oleg R. Latypov , Michael G. Shlyapnikov , Maria A. Gorshkova , Eva N. Titova , Natalia N. Vlasova , Alexey V. Lipkin , Alexey N. Fedorov , Igor E. Granovsky

{"title":"A method for producing protease pS273R of the African swine fever virus","authors":"Danil S. Kalinin , Sergey G. Mayorov , Marina Yu. Zemskova , Oleg R. Latypov , Michael G. Shlyapnikov , Maria A. Gorshkova , Eva N. Titova , Natalia N. Vlasova , Alexey V. Lipkin , Alexey N. Fedorov , Igor E. Granovsky","doi":"10.1016/j.jviromet.2024.115037","DOIUrl":"10.1016/j.jviromet.2024.115037","url":null,"abstract":"<div><div>The pS273R protease of the African swine fever virus (ASFV) is responsible for the processing of the viral polyproteins pp220 and pp62, precursors of the internal capsid of the virus. The protease is essential for a productive viral infection and is an attractive target for antiviral therapy. This work presents a method for the production of pS273R in <em>E. coli</em> cells by fusing the protease with the SlyD chaperone. The chimeric protein pS273R protease, during expression, is formed in a soluble form possessing enzymatic activity. Subsequently, pS273R separates from SlyD through autocatalytic cleavage at the TEV protease site <em>in vivo</em>. This work devised a straightforward protocol for chromatographic purification, resulting in the production of a highly purified viral protease. Additionally, we suggest using a fluorescence method to assess the activity of pS273R. This method is predicated on a shift in the chimeric protein thioredoxin-EGFP's electrophoretic mobility following its protease cleavage. It was shown that thioredoxin-EGFP substrate is effectively and selectively cleaved by the pS273R protease, even in complex protein mixtures such as mammalian cell lysates.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115037"},"PeriodicalIF":2.2,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142328095","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development and validation of a quantitative PCR assay for detection of Sulawesi tortoise adenovirus 开发和验证用于检测苏拉威西陆龟腺病毒的定量 PCR 检测方法。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-21 DOI: 10.1016/j.jviromet.2024.115033

Zachary C. Ready , Laura Adamovicz , Maris Daleo , Amber Simmons , Matthew C. Allender

{"title":"Development and validation of a quantitative PCR assay for detection of Sulawesi tortoise adenovirus","authors":"Zachary C. Ready , Laura Adamovicz , Maris Daleo , Amber Simmons , Matthew C. Allender","doi":"10.1016/j.jviromet.2024.115033","DOIUrl":"10.1016/j.jviromet.2024.115033","url":null,"abstract":"<div><div>In 2007, a mortality event involving over 100 Sulawesi tortoises (<em>Indotestudo forsteni</em>), two Impressed tortoises (<em>Manouria impress</em>) and a critically endangered Burmese star tortoise (<em>Geochelone platynota</em>) was attributed to Sulawesi tortoise adenovirus (STADV; genus <em>Siadenovirus</em>). We developed a TaqMan quantitative PCR assay targeting the DNA polymerase gene of STADV for use in clinical diagnosis and epidemiologic surveillance. This assay failed to amplify five closely-related chelonian adenoviruses, indicating high analytical specificity. The assay performed with high efficiency (slope = −3.337; R<sup>2</sup> = 0.999) and high inter- and intra-assay repeatability (coefficient of variation <1.36 % at all standard curve dilutions). Dynamic range included 1.00 × 10<sup>7</sup> to 1.00 × 10<sup>1</sup> target copies per reaction and limit of detection was 10<sup>1</sup> target copies per reaction, though 10<sup>0</sup> target copies per reaction were intermittently detected. This qPCR assay provides a valuable diagnostic tool for characterization of STADV epidemiology, including potential identification of the North American reservoir host.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115033"},"PeriodicalIF":2.2,"publicationDate":"2024-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142308002","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Reducing amplification cycles to improve the coverage of influenza A virus genome sequencing in heterosubtypic co-infection 减少扩增周期，提高甲型流感病毒基因组测序在异种亚型共同感染中的覆盖率。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-20 DOI: 10.1016/j.jviromet.2024.115036

Yijie Zhang , Wenhua Kong , Yixuan Wu , Zhi Chen , Xiang Zhao , Manqing Liu

引用次数: 0

Performance of an in-house multiplex PCR assay for HIV-1 drug resistance testing – A cheaper alternative 用于 HIV-1 耐药性检测的内部多重 PCR 分析法的性能--一种更便宜的替代方法。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-18 DOI: 10.1016/j.jviromet.2024.115034

Tumelo L. Fortuin , Paballo Nkone , Allison Glass , Raquel Viana , Keitumetse Moeng , Shayne Loubser , Caroline T. Tiemessen , Simnikiwe H. Mayaphi

{"title":"Performance of an in-house multiplex PCR assay for HIV-1 drug resistance testing – A cheaper alternative","authors":"Tumelo L. Fortuin , Paballo Nkone , Allison Glass , Raquel Viana , Keitumetse Moeng , Shayne Loubser , Caroline T. Tiemessen , Simnikiwe H. Mayaphi","doi":"10.1016/j.jviromet.2024.115034","DOIUrl":"10.1016/j.jviromet.2024.115034","url":null,"abstract":"<div><h3>Background</h3><div>Currently, most HIV drug resistance PCR assays amplify the protease-reverse transcriptase (PR-RT) fragment separately from the integrase (IN) fragment. The aim of this study was to develop a multiplex PCR assay that simultaneously amplifies PR-RT and IN fragments for HIV-1 drug-resistance testing.</div></div><div><h3>Methods</h3><div>The in-house multiplex PCR assay was evaluated on extracted total nucleic acids obtained from the National Health Laboratory Service (NHLS) and Lancet laboratories. Sanger sequencing was performed on amplicons, and HIV-1 drug-resistance mutations (DRMs) were assessed using HIV Stanford drug resistance database.</div></div><div><h3>Results</h3><div>This study tested 59 patient samples with known HIV-1 viral load and DRM results; 41 from Lancet and 18 from NHLS. In-house multiplex PCR assay detected one or both fragments in most samples but had higher sensitivity for detection of IN fragment (93.2 %) compared to PR-RT fragment (83.1 %). There was 100 % concordance between Lancet assay versus in-house assay sequence data for IN DRMs, but lower concordance with PR-RT (87.0 %). The in-house multiplex PCR assay’s precision and reproducibility analysis showed ≥99.9 % sequence similarity and yielded similar DRM results for both PR-RT and IN fragments.</div></div><div><h3>Conclusions</h3><div>The in-house multiplex PCR assay demonstrated satisfactory performance and higher sensitivity for IN fragment amplification. This could be a cost-effective method for HIV-1 drug resistance testing as both PR-RT and IN fragments are successfully amplified in one reaction in most samples.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115034"},"PeriodicalIF":2.2,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0166093424001587/pdfft?md5=1d8773b905f46d2f79c1de4d12dac17f&pid=1-s2.0-S0166093424001587-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142290211","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Rapid detection of bat coronaviruses from fecal samples using loop-mediated isothermal amplification assay in the field 在野外使用环介导等温扩增分析法从粪便样本中快速检测蝙蝠冠状病毒

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-17 DOI: 10.1016/j.jviromet.2024.115035

Undarmaa Tsengel , Tzong-Yuan Wu , Yi-Ning Chen

{"title":"Rapid detection of bat coronaviruses from fecal samples using loop-mediated isothermal amplification assay in the field","authors":"Undarmaa Tsengel , Tzong-Yuan Wu , Yi-Ning Chen","doi":"10.1016/j.jviromet.2024.115035","DOIUrl":"10.1016/j.jviromet.2024.115035","url":null,"abstract":"<div><p>The global impact of the COVID-19 pandemic has emphasized the critical need for effective viral diagnostics. Although polymerase chain reaction (PCR) is a well-established nucleotide amplification technique, its limitations, such as the need for expensive equipment and skilled technicians, have led to the exploration of alternative methods, including loop-mediated isothermal amplification (LAMP). Bats, as a crucial natural reservoir of coronaviruses (CoVs), particularly <em>Scotophilus</em> bat coronavirus 512 (<em>Scotophilus</em> bat-CoV 512) prevalent among Taiwan’s bat population, are the focus of this study. We aimed to detect <em>Scotophilus</em> bat-CoV 512 from bats in field conditions using loop-mediated isothermal amplification (LAMP) assay for on-site detection. Therefore, our study delves into the specificity of the LAMP reaction, emphasizing the careful design of primers to prevent false positive results. A cross reactivity and primer specificity test involving seven different microorganisms, including closely related bat CoVs and two bacterial species typically found in feces, revealed that the LAMP assay uniquely detected <em>Scotophilus</em> bat-CoV 512. The developed colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was optimized for the primers targeting nucleocapsid (N) gene, and the sensitivity test revealed a detection limit of 2.4 × 10<sup>3</sup> copies/µL. Our findings indicate the potential of the RT-LAMP assay for on-site detection in the field and subsequent laboratory analysis for comprehensive sampling and further research on bat CoV isolation. The surveillance and monitoring of bat CoVs contribute substantially to mitigating human threats, particularly concerning the emergence of new pandemic variants.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115035"},"PeriodicalIF":2.2,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142271062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Establishment of a reverse genetics system for virulent systemic feline calicivirus using circular polymerase extension reaction 利用环形聚合酶延伸反应建立毒性系统性猫科动物卡里科病毒的反向遗传学系统

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-08 DOI: 10.1016/j.jviromet.2024.115031

Xiao Wang, Da Zhang, Aoxing Tang, Miao Zhang, Shiqiang Zhu, Yingqi Zhu, Bo Li, Chunchun Meng, Chuanfeng Li, Jie Zhu, Guangqing Liu

{"title":"Establishment of a reverse genetics system for virulent systemic feline calicivirus using circular polymerase extension reaction","authors":"Xiao Wang, Da Zhang, Aoxing Tang, Miao Zhang, Shiqiang Zhu, Yingqi Zhu, Bo Li, Chunchun Meng, Chuanfeng Li, Jie Zhu, Guangqing Liu","doi":"10.1016/j.jviromet.2024.115031","DOIUrl":"10.1016/j.jviromet.2024.115031","url":null,"abstract":"<div><h3>Summary</h3><p>Feline caliciviruses can cause oral and upper respiratory tract infections in cats. However, a virulent and systemic feline calicivirus (VS-FCV) variant implicated in multisystem lesions and death in cats has emerged recently. To date, the mechanism underlying virulence variations in VS-FCV remains unclear. The aim of the present study was to provide a tool for exploring genetic variation in VS-FCV, by constructing an infectious clone of VS-FCV SH/2014. First, a full-length cDNA molecular clone of VS-FCV SH/2014 strain, which contains an Xba I recognition site generated by mutating one base (A→T) as a genetic marker, was constructed using the circular polymerase extension reaction (CPER) method. Second, the full-length cDNA clone was introduced into Crandell-Rees feline kidney cells using liposomes to rescue recombinant VS-FCV SH/2014 (rVS-FCV SH/2014). Third, the rescued viruses were identified by real-time PCR, immunofluorescence assay, western blotting, and electron microscopy. The full-length cDNA molecular clone of the VS-FCV SH/2014 strain was successfully constructed and that rVS-FCV SH/2014 could be rescued efficiently. rVS-FCV SH/2014 had the expected genetic markers and morphology and growth characteristics similar to those of the parental virus. The reverse genetics system provides a research platform for future studies on VS-FCV genetic variation and pathogenesis.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115031"},"PeriodicalIF":2.2,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142230797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Real-time quantitative reverse transcription PCR assay for the detection of Nuomin virus – An emerging tick-borne virus 实时定量反转录 PCR 检测 Nuomin 病毒--一种新出现的蜱传病毒

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-07 DOI: 10.1016/j.jviromet.2024.115032

Kairao Hu , Tingting Liu , Wenbo Xu , Ziyan Liu , Zhedong Wang , Jun Ma , Quan Liu

{"title":"Real-time quantitative reverse transcription PCR assay for the detection of Nuomin virus – An emerging tick-borne virus","authors":"Kairao Hu , Tingting Liu , Wenbo Xu , Ziyan Liu , Zhedong Wang , Jun Ma , Quan Liu","doi":"10.1016/j.jviromet.2024.115032","DOIUrl":"10.1016/j.jviromet.2024.115032","url":null,"abstract":"<div><p>Nuomin virus (NOMV), an emerging tick-borne virus (TBVs) identified in 2020, has been associated with fever, headache, and potential liver dysfunction in infected individuals. This study presents a novel TaqMan real-time quantitative PCR method designed for the rapid, sensitive, and specific detection of NOMV, facilitating early diagnosis. Utilizing Beacon Designer software 8.0, we optimized the PCR assay including the development of primers and probes to precisely target the conserved region of the NOMV genome, followed by optimization of primer and probe concentrations and annealing temperature. The resulting assay demonstrated robust performance, with standard curve represented by the equation y=−3.29x+39.42, a high correlation coefficient (R<sup>2</sup> = 0.995) and an efficiency 99.53 %. Importantly, the method exhibited exceptional specificity, which did not yield cross-reacting signals from other TBVs, including Songling virus (SGLV), Beiji virus (BJNV), tick-borne encephalitis virus (TBEV), Yezo virus (YEZV), Alongshan virus (ALSV), and severe fever with thrombocytopenia syndrome bunyavirus (SFTSV). The assay’s detection limit was remarkably low, reaching 10 copies/μL, representing a 100-fold increase compared to semi-nested RT-PCR. Additionally, it demonstrated excellent repeatability, with coefficients of variation for intra- and inter-group tests consistently below 3 %. Clinical evaluations confirmed the assay’s superior performance, highlighting its high specificity, sensitivity, and reproducibility for NOMV detection. In conclusion, the method developed in this study provides a valuable tool to support timely management of NOMV infections, with significant implications for clinical practice.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115032"},"PeriodicalIF":2.2,"publicationDate":"2024-09-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142167739","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Efficient and accurate BmNPV bacmid editing system by two-step golden gate assembly 通过两步金门组装实现高效、精确的 BmNPV Bacmid 编辑系统。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-06 DOI: 10.1016/j.jviromet.2024.115029

Takeru Ebihara , Misaki Shibuya , Ayaka Yamaguchi , Masato Hino , Jae Man Lee , Takahiro Kusakabe , Hiroaki Mon

{"title":"Efficient and accurate BmNPV bacmid editing system by two-step golden gate assembly","authors":"Takeru Ebihara , Misaki Shibuya , Ayaka Yamaguchi , Masato Hino , Jae Man Lee , Takahiro Kusakabe , Hiroaki Mon","doi":"10.1016/j.jviromet.2024.115029","DOIUrl":"10.1016/j.jviromet.2024.115029","url":null,"abstract":"<div><p>The silkworm-baculovirus expression vector system (silkworm-BEVS), using <em>Bombyx mori</em> nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130 kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme <em>Bsa</em>I. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme <em>Paq</em>CI with a high efficiency of 97.2 %. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115029"},"PeriodicalIF":2.2,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0166093424001538/pdfft?md5=aa4f72af6849555c79b8781186ba48d0&pid=1-s2.0-S0166093424001538-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142145912","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Carboxy-PEG-thiol functionalized gold nanoparticle conjugates for the detection of SARS-CoV-2: Detection tools and analytical method development 用于检测 SARS-CoV-2 的羧基-聚乙二醇-硫醇功能化金纳米粒子共轭物：检测工具和分析方法开发。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-09-03 DOI: 10.1016/j.jviromet.2024.115028

Lerato Hlekelele , Katlego Setshedi , Vusani Mandiwana , Lonji Kalombo , Yolandy Lemmer , Vongani Chauke , Arjun Maity

{"title":"Carboxy-PEG-thiol functionalized gold nanoparticle conjugates for the detection of SARS-CoV-2: Detection tools and analytical method development","authors":"Lerato Hlekelele , Katlego Setshedi , Vusani Mandiwana , Lonji Kalombo , Yolandy Lemmer , Vongani Chauke , Arjun Maity","doi":"10.1016/j.jviromet.2024.115028","DOIUrl":"10.1016/j.jviromet.2024.115028","url":null,"abstract":"<div><p>Addressing the need for accessible SARS-CoV-2 testing, carboxy-PEG 12-thiol functionalized gold nanoparticles conjugates were developed for rapid point-of-care (POC) detection against SARS-CoV-2 spike protein, pseudo-SARS-CoV-2, and authentic Beta SARS-CoV-2 virus particles. These conjugates leverage gold nanoparticles (AuNPs) as signal transducers, cross-linked to either angiotensin-converting enzyme 2 (ACE2) or SARS-CoV-2 spike protein receptor-binding domain (RBD) antibodies as bioreceptors and showed a distinct color shift from pink to blue. To assess their POC feasibility, the conjugates were integrated into facemasks and breathalyzers, wherein aerosolized SARS-CoV-2 antigens were successfully detected, producing a color change within 10 and 30 minutes for the breathalyzer and facemask prototypes, respectively. Furthermore, we explored quantitative analysis using varying concentrations of SARS-CoV-2 spike protein. Both conjugates demonstrated a linear relationship between blue color intensity and virus concentration, with linear ranges of 0.08–0.6 ng/mL and 0.04–0.5 ng/mL, respectively. Low limits of detection and quantification were also achieved. They exhibited specificity, responding solely to SARS-CoV-2 even in complex matrices containing diverse proteins, including the SARS-CoV-1 spike protein. Precision tests yielded coefficient of variations below 2 %, showcasing their remarkable reproducibility. This work presents a promising approach for rapid, sensitive, and specific POC detection of SARS-CoV-2 paving the way for improved pandemic response and management.</p></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"330 ","pages":"Article 115028"},"PeriodicalIF":2.2,"publicationDate":"2024-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0