RESUMEN
Human enteroviruses (HEV) can cause a range of diseases from mild to potentially life-threatening. Identification and genotyping of HEV are crucial for disease management. Existing typing methods, however, have inherent limitations. Developing alternative methods to detect HEV with more virus types, high accuracy, and sensitivity in an accessible manner presents a technological and analytical challenge. Here, a sequence-specific nanoparticle barcode (SSNB) method is presented for simultaneous detection of 10 HEV types. This method significantly increases sensitivity, enhancing detection by 10-106 times over the traditional multiplex hybrid genotyping (MHG) method, by resolving cross-interference between the multiple primer sets. Furthermore, the SSNB method demonstrates a 100% specificity in accurately distinguishing between 10 different HEV types and other prevalent clinical viruses. In an analysis of 70 clinical throat swab samples, the SSNB method shows slightly higher detection rate for positive samples (50%) compared to the RT-PCR method (48.6%). Additionally, further assessment of the typing accuracy for samples identified as positive by SSNB using sequencing method reveals a concordance rate of 100%. The combined high sensitivity and specificity level of the methodology, together with the capability for multiple type analysis and compatibility with clinical workflow, make this approach a promising tool for clinical settings.
Asunto(s)
Infecciones por Enterovirus , Enterovirus , Nanopartículas , Humanos , Nanopartículas/química , Infecciones por Enterovirus/virología , Infecciones por Enterovirus/diagnóstico , Enterovirus/genética , Enterovirus/clasificación , Enterovirus/aislamiento & purificación , Código de Barras del ADN Taxonómico/métodos , Sensibilidad y Especificidad , Técnicas de Genotipaje/métodos , Genotipo , ARN Viral/genéticaRESUMEN
SARS-CoV-2 variants of concern (VOCs) contain several single-nucleotide variants (SNVs) at key sites in the receptor-binding region (RBD) that enhance infectivity and transmission, as well as cause immune escape, resulting in an aggravation of the coronavirus disease 2019 (COVID-19) pandemic. Emerging VOCs have sparked the need for a diagnostic method capable of simultaneously monitoring these SNVs. To date, no highly sensitive, efficient clinical tool exists to monitor SNVs simultaneously. Here, an encodable multiplex microsphere-phase amplification (MMPA) sensing platform that combines primer-coded microsphere technology with dual fluorescence decoding strategy to detect SARS-CoV-2 RNA and simultaneously identify 10 key SNVs in the RBD. MMPA limits the amplification refractory mutation system PCR (ARMS-PCR) reaction for specific target sequence to the surface of a microsphere with specific fluorescence coding. This effectively solves the problem of non-specific amplification among primers and probes in multiplex PCR. For signal detection, specific fluorescence codes inside microspheres are used to determine the corresponding relationship between the microspheres and the SNV sites, while the report probes hybridized with PCR products are used to detect the microsphere amplification intensity. The MMPA platform offers a lower SARS-CoV-2 RNA detection limit of 28 copies/reaction, the ability to detect a respiratory pathogen panel without cross-reactivity, and a SNV analysis accuracy level comparable to that of sequencing. Moreover, this super-multiple parallel SNVs detection method enables a timely updating of the panel of detected SNVs that accompanies changing VOCs, and presents a clinical availability that traditional sequencing methods do not.
Asunto(s)
Técnicas Biosensibles , COVID-19 , COVID-19/diagnóstico , Humanos , Microesferas , Reacción en Cadena de la Polimerasa Multiplex , Mutación , ARN Viral/genética , SARS-CoV-2/genéticaRESUMEN
Convective PCR (CPCR) is an isothermal nucleic acid amplification technology; however, natural convection exhibits a chaotic and multiplex flow state, resulting in low amplification efficiency and specificity. We placed a polycarbonate strip (p-strip) inside reaction tubes to induce circumfluence by blocking the inner ring that originally allowed fluid to flow at suboptimal temperatures. Moreover, we constructed a dual-temperature instrument to provide appropriate denaturing and annealing zones for CPCR. Tubes containing p-strips exhibited significantly improved efficiency, sensitivity and specificity. For real-time detection, the variation coefficients of three replicates having the same concentrations were less than 2% in more than half of the cases, indicating improved CPCR amplification and potential as a commercial on-site nucleic acid diagnosis tool.
Asunto(s)
Ácidos Nucleicos/genética , Pruebas en el Punto de Atención , Reacción en Cadena de la Polimerasa/métodos , Convección , Infecciones por Coxsackievirus/virología , Citomegalovirus/genética , Infecciones por Citomegalovirus/virología , Enterovirus/genética , Diseño de Equipo , Humanos , Pruebas en el Punto de Atención/economía , Reacción en Cadena de la Polimerasa/economía , Reacción en Cadena de la Polimerasa/instrumentación , TemperaturaRESUMEN
The convective polymerase chain reaction (CCPCR) uses the principle of thermal convection to allow the reagent to flow in the test tube and achieve the purpose of amplification by the temperature difference between the upper and lower portions of the test tube. In order to detect the amplification effect in real time, we added a fluorophore to the reagent system to reflect the amplification in real time through the intensity of fluorescence. The experimental results show that the fluorescence curve conforms to the S-type trend of the amplification curve, but there is a certain jitter condition due to the instability of the thermal convection, which is not conducive to the calculation of the cycle threshold (CT value). In order to solve this problem, this paper uses the dynamic method, using the double S-type function model to fit the curve, so that the fluorescence curve is smooth and the initial concentration of the nucleic acid can be deduced better to achieve the quantitative purpose based on the curve. At the same time, the PSO+ algorithm is used to solve the double s-type function parameters, that is, particle swarm optimization (PSO) algorithm combined with Levenberg-Marquardt, Newton-CG and other algorithms for curve fitting. The proposed method effectively overcoms PSO randomness and the shortcoming of traditional algorithms such as Levenberg-Marquardt and Newton-CG which are easy to fall into the local optimal solution. The R 2 of the data fitting result can reach 0.999 8. This study is of guiding significance for the future quantitative detection of real-time fluorescent heat convection amplification.
Asunto(s)
Algoritmos , Fluorescencia , Reacción en Cadena de la Polimerasa , Colorantes FluorescentesRESUMEN
Zika virus (ZIKV), dengue virus (DENV), chikungunya virus (CHIKV) and yellow fever virus (YFV) share the same mosquito vectors and have similar clinical manifestations early stage of infection. Therefore, simultaneously differentiating these viruses from each other is necessary. We developed a multiplex real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay for the differentiation of these four viruses in a single tube. The linear range was established by regression analysis, and the R2 value for each virus was ≥0.98, and the 95% lower limit of detection for each virus was as follows (copies/reaction): ZIKV-Asian, 9; ZIKV-Africa, 15; CHIKV, 11; DENV-1, 19; DENV-2, 13; DENV-3, 24; DENV-4, 36; and YFV, 17. Meanwhile, our multiplex real-time RT-PCR has a good consistency with the commercial singleplex assay. In summary, the developed assay can be effectively used for the diagnosis of ZIKV, DENV, CHIKV, and YFV infections.
Asunto(s)
Fiebre Chikungunya/diagnóstico , Dengue/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Fiebre Amarilla/diagnóstico , Infección por el Virus Zika/diagnóstico , África , Fiebre Chikungunya/virología , Virus Chikungunya/genética , Virus Chikungunya/aislamiento & purificación , Dengue/virología , Virus del Dengue/genética , Virus del Dengue/aislamiento & purificación , Humanos , Técnicas de Diagnóstico Molecular/métodos , Factores de Tiempo , Fiebre Amarilla/virología , Virus de la Fiebre Amarilla/genética , Virus de la Fiebre Amarilla/aislamiento & purificación , Virus Zika/genética , Virus Zika/aislamiento & purificación , Infección por el Virus Zika/virologíaRESUMEN
Enterovirus (EV)-A71 and Coxsackievirus (CV)-A16 have historically been the major pathogens of hand, foot, and mouth disease (HMFD) in China; however, CV-A6ï¼ which had previously received little attention, became the predominant pathogen in 2013, and has remained one of the common pathogens since then. In this work, we conducted a molecular epidemiology study of CV-A6-associated HFMD in Xiamen from 2009 to 2015. The data showed CV-A6 pandemics had a certain periodicity rather than occurring randomly. Evolution analysis based on near-complete VP1 nucleotide sequences showed subgenotype D5 lineage 4 strains account for the persistent outbreak of CV-A6-associated HFMD in China since 2013. Alignment analysis revealed eight candidate amino acid substitutions in VP1, which may provide useful information for the research of CV-A6 virulence enhancement. This study contributed to elucidating the circulation patterns and genetic characteristics of CV-A6 in China; however, further surveillance and intervention in CV-A6 epidemics is recommended.
Asunto(s)
Brotes de Enfermedades , Enterovirus/clasificación , Enterovirus/genética , Genotipo , Enfermedad de Boca, Mano y Pie/epidemiología , Enfermedad de Boca, Mano y Pie/virología , China/epidemiología , Enterovirus/aislamiento & purificación , Epidemiología Molecular , Análisis de Secuencia de ADN , Proteínas Estructurales Virales/genéticaRESUMEN
BACKGROUND: Morbidity and mortality from influenza A (Flu A) have increased in recent years. Timely diagnosis and management are critical for disease control. Therefore, the development of a rapid, accurate, and portable analytical method for on-site analysis is imperative. OBJECTIVES: The aim of this work was to develop a rapid, on-site, automated assay for the detection of Flu A and to evaluate the assay. METHODS: A handheld instrument (TD-01) based on capillary convective polymerase chain reaction (PCR) was developed for rapid on-site detection of Flu A. Since a previous version of the instrument, an automated motion mechanism has been introduced to TD-01 to achieve RNA automated testing. The primers and probe used for Flu A detection were designed according to the Flu A gene sequence of matrix proteins. Finally, we evaluated the detection spectra, sensitivity, specificity, and diagnostic performance of the assay. RESULTS: The TD-01 was able to successfully automatically detect Flu A RNA within 30 min. Results for serially diluted viruses indicated that the lower limit of detection for Flu A was 0.1 TCID50/ml (50% tissue culture infective dose). After evaluating known virus stocks, including 15 strains of Flu A, four strains of Flu B, and two strains of respiratory syncytial virus (RSV), the assay had a favorable detection spectrum and no obvious cross-reactivity. Method verification based on 554 clinical samples indicated that the sensitivity and specificity of TD-01 were 98.30% (231/235) and 98.75% (315/319), respectively. CONCLUSIONS: The results indicate that Flu A detection by TD-01 is particularly suitable for on-site testing and has the potential for application in point-of-care testing.