RESUMO
Rapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal reverse transcription strand invasion based amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 min. The Egoo Health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0-98.4% and 96.6-98.2% respectively.
Assuntos
COVID-19/diagnóstico , COVID-19/epidemiologia , Desenho de Equipamento , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Diagnóstico Molecular/métodos , Pandemias/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase Reversa/instrumentação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , SARS-CoV-2/genética , COVID-19/virologia , Telefone Celular , Humanos , Aplicativos Móveis , Orofaringe/virologia , Testes Imediatos , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
Zika virus has only recently gained attention due to recent large outbreaks worldwide. An easy to use nucleic acid amplification test could play an important role in the early detection of the infection and patient management. Here, we report a rapid and robust isothermal nucleic acid amplification assay for the detection of Zika virus. The method is cost-effective and compatible with portable instrumentation, enabling near patient testing and field use.
Assuntos
Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecção por Zika virus/diagnóstico , Zika virus/isolamento & purificação , Análise Custo-Benefício , Humanos , Fatores de Tempo , Zika virus/genéticaRESUMO
Rapid and accurate diagnosis of influenza viruses plays an important role in infection control, as well as in preventing the misuse of antibiotics. Isothermal nucleic acid amplification methods offer significant advantages over the polymerase chain reaction (PCR), since they are more rapid and do not require the sophisticated instruments needed for thermal cycling. We previously described a novel isothermal nucleic acid amplification method, 'Strand Invasion Based Amplification' (SIBA®), with high analytical sensitivity and specificity, for the detection of DNA. In this study, we describe the development of a variant of the SIBA method, namely, reverse transcription SIBA (RT-SIBA), for the rapid detection of viral RNA targets. The RT-SIBA method includes a reverse transcriptase enzyme that allows one-step reverse transcription of RNA to complementary DNA (cDNA) and simultaneous amplification and detection of the cDNA by SIBA under isothermal reaction conditions. The RT-SIBA method was found to be more sensitive than PCR for the detection of influenza A and B and could detect 100 copies of influenza RNA within 15 min. The development of RT-SIBA will enable rapid and accurate diagnosis of viral RNA targets within point-of-care or central laboratory settings.
Assuntos
Vírus da Influenza A/isolamento & purificação , Vírus da Influenza B/isolamento & purificação , Influenza Humana/diagnóstico , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Transcrição Reversa , Primers do DNA , Humanos , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Sistemas Automatizados de Assistência Junto ao Leito , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
We developed a modified flagellar type III secretion apparatus to secrete heterologous polypeptides into the growth medium of Escherichia coli. The secretion was facilitated by fusing the 173-bp untranslated DNA fragment upstream of the gene fliC (encoding flagellin) as well as a transcriptional terminator from fliC, into the gene encoding the polypeptide of interest. The polypeptides secreted into the growth medium at concentrations ranging from 1 to 15 mg/l were from Campylobacter jejuni (262 residues in length), Streptococcus pneumoniae (434 residues), Staphylococcus aureus (115 residues), and N-terminal FliC hybrid proteins, for example, the eukaryotic green fluorescent protein (238 residues). The expressed proteins represented >50% of total secreted protein. Previously reported protein yields from extracellular secretion of foreign proteins in E. coli have been low, approximately 100 microg/l. The strengths of our method are the concentration and purity of the secreted proteins and its versatility with regard to the proteins' length and origin.