scholarly journals Ultrasensitive RNA biosensors for SARS-CoV-2 detection in a simple color and luminescence assay

2021 ◽  
Author(s):  
Anirudh Chakravarthy ◽  
Anirudh K N ◽  
Geen George ◽  
Shyamsundar Ranganathan ◽  
Nishan Shettigar ◽  
...  
Keyword(s):  
Cell Phone ◽  
Rna Amplification ◽  
Reporter Protein ◽  
Human Patient ◽  
Rna Detection ◽  
Conformational Switch ◽  
Diagnostic Strategies ◽  
Highly Sensitive ◽  
Luminescence Assay ◽  
Biosensor Response

The COVID-19 pandemic underlines the need for versatile diagnostic strategies. Here, we have designed and developed toehold RNA-based sensors for direct and ultrasensitive SARS-CoV-2 RNA detection. In our assay, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter-protein e.g. LacZ or Nano-lantern that is easily detected using color/luminescence. This response can be visualized by the human eye, or a simple cell phone camera as well as quantified using a spectrophotometer/luminometer. By optimizing RNA-amplification and biosensor-design, we have generated a highly-sensitive diagnostic assay; with sensitivity down to attomolar (100 copies of) SARS-CoV-2 RNA. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) efficiently detects the presence of viral RNA in human patient samples, with clear distinction from samples designated negative for the virus. The biosensor response correlates well with Ct values from RT-qPCR tests and thus presents a powerful and easily accessible strategy for detecting Covid infection.

scholarly journals Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay

2021 ◽  
Vol 4 (12) ◽  
pp. e202101213
Author(s):  
Anirudh Chakravarthy ◽  
Anirudh Nandakumar ◽  
Geen George ◽  
Shyamsundar Ranganathan ◽  
Suchitta Umashankar ◽  
...  
Keyword(s):  
Viral Evolution ◽  
Viral Rna ◽  
Diagnostic Assay ◽  
Rna Amplification ◽  
Reporter Protein ◽  
Conformational Switch ◽  
Highly Sensitive ◽  
Luminescence Assay ◽  
Bright Color ◽  
Multiple Variants

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA–based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

scholarly journals Improved Production of Heterologous Proteins by a Glucose Repression-Defective Mutant of Kluyveromyces lactis

2004 ◽  
Vol 70 (5) ◽  
pp. 2632-2638 ◽  
Author(s):  
Claudia Donnini ◽  
Francesca Farina ◽  
Barbara Neglia ◽  
Maria Concetta Compagno ◽  
Daniela Uccelletti ◽  
...  
Keyword(s):  
Glucose Repression ◽  
Kluyveromyces Lactis ◽  
Kinase Domain ◽  
Arxula Adeninivorans ◽  
Nucleotide Position ◽  
Heterologous Proteins ◽  
Reporter Protein ◽  
Gene Encoding ◽  
Defective Mutant ◽  
Highly Sensitive

ABSTRACT The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1β compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.

scholarly journals Epidemiology and Clinical Characteristics of COVID-19

2020 ◽  
Vol 23 (4) ◽  
pp. 268-271 ◽  
Author(s):  
Xiaoyi Huang ◽  
Fengxiang Wei ◽  
Liang Hu ◽  
Lijuan Wen ◽  
Ken Chen
Keyword(s):  
Clinical Manifestations ◽  
Supportive Therapy ◽  
Epidemiological Studies ◽  
World Health ◽  
Severe Illness ◽  
Rna Detection ◽  
Highly Sensitive ◽  
Novel Coronavirus ◽  
Health Organization ◽  
Insight Into

Since December 2019, there has been an outbreak of a novel coronavirus (COVID-19) infection in Wuhan, China. Meanwhile, the outbreak also drew attention and concern from the World Health Organization (WHO). COVID-19 is another human infectious disease caused by coronavirus. The transmission of COVID-19 is potent and the infection rate is fast. Since there is no specific drug for COVID-19, the treatment is mainly symptomatic supportive therapy. In addition, it should be pointed out that patients with severe illness need more aggressive treatment and meticulous care. Recently, accurate RNA detection has been decisive for the diagnosis of COVID-19. The development of highly sensitive RT-PCR has facilitated epidemiological studies that provide insight into the prevalence, seasonality, clinical manifestations and course of COVID-19 infection. In this review, we summarize the epidemiology and characteristics of COVID-19.

Highly sensitive hepatitis C virus RNA detection assays for decision of treatment (dis)continuation in patients with chronic hepatitis C

2005 ◽  
Vol 42 (4) ◽  
pp. 605-606 ◽  
Author(s):  
Ulrike Mihm ◽  
Wolf-Peter Hofmann ◽  
Bernd Kronenberger ◽  
Michael von Wagner ◽  
Stefan Zeuzem ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Hepatitis C Virus Rna ◽  
Rna Detection ◽  
Virus Rna ◽  
Highly Sensitive

scholarly journals Immunoproteomic analysis of Borrelia miyamotoi for the identification of serodiagnostic antigens

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Emma K. Harris ◽  
Marisa R. Harton ◽  
Maria Angela de Mello Marques ◽  
John T. Belisle ◽  
Claudia R. Molins ◽  
...  
Keyword(s):  
Lyme Disease ◽  
Specific Antigen ◽  
Disease Patient ◽  
Borrelia Miyamotoi ◽  
Serum Samples ◽  
Human Patient ◽  
Highly Sensitive ◽  
Associated Proteins ◽  
Multiple Variable ◽  
Health Significance

AbstractThe tick-borne spirochete, Borrelia miyamotoi, is an emerging pathogen of public health significance. Current B. miyamotoi serodiagnostic testing depends on reactivity against GlpQ which is not highly sensitive on acute phase serum samples. Additionally, anti-B. miyamotoi antibodies can cross-react with C6 antigen testing for B. burgdorferi, the causative agent of Lyme disease, underscoring the need for improved serological assays that produce accurate diagnostic results. We performed an immunoproteomics analysis of B. miyamotoi proteins to identify novel serodiagnostic antigens. Sera from mice infected with B. miyamotoi by subcutaneous inoculation or tick bite were collected for immunoblotting against B. miyamotoi membrane-associated proteins separated by 2-dimensional electrophoresis (2DE). In total, 88 proteins in 40 2DE immunoreactive spots were identified via mass spectrometry. Multiple variable large proteins (Vlps) and a putative lipoprotein were among those identified and analyzed. Reactivity of anti-B. miyamotoi sera against recombinant Vlps and the putative lipoprotein confirmed their immunogenicity. Mouse anti-B. burgdorferi serum was cross-reactive to all recombinant Vlps, but not against the putative lipoprotein by IgG. Furthermore, antibodies against the recombinant putative lipoprotein were present in serum from a B. miyamotoi-infected human patient, but not a Lyme disease patient. Results presented here provide a comprehensive profile of B. miyamotoi antigens that induce the host immune response and identify a putative lipoprotein as a potentially specific antigen for B. miyamotoi serodetection.

l-DNA-tagged fluorescence in situ hybridization for highly sensitive imaging of RNAs in single cells

2020 ◽  
Vol 18 (40) ◽  
pp. 8084-8088
Author(s):  
Motoyuki Ogata ◽  
Gosuke Hayashi ◽  
Anri Ichiu ◽  
Akimitsu Okamoto
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Single Cells ◽  
Rna Detection ◽  
Pcr Product ◽  
Highly Sensitive

l-DNA tagged FISH (LT-FISH), including two-step hybridization processes with a l–d chimera oligonucleotide and a fluorescence-labeled PCR product tethering a l-DNA tag, has realized sensitive RNA detection in fixed cells.

scholarly journals Strategies of RT-PCR-based assay design and surveillance of SARS-CoV-2

2020 ◽  
Author(s):  
NA Kuznetsova ◽  
AA Pochtovyy ◽  
MA Nikiforova ◽  
VA Guschin
Keyword(s):  
Transportation Systems ◽  
Tourism Industry ◽  
Molecular Techniques ◽  
Clinical Samples ◽  
The Novel ◽  
Rt Pcr ◽  
Rna Detection ◽  
Highly Sensitive ◽  
Current Outbreak ◽  
Assay Design

High population density in the cities with bustling transportation systems and a thriving tourism industry can promote the global spread of a viral infection in a matter of days. The novel SARS-CoV-2 coronavirus has already infected over 2,000,000 people worldwide and caused upwards of 156,000 deaths. One of the factors driving the rapid unfolding of the pandemic is the absence of diagnostic tests for SARS-CoV-2 detection. Molecular techniques allow SARS-CoV-2 RNA to be quickly detected in clinical samples, aiding the differential diagnosis in severely ill patients and facilitating identification of asymptomatic carriers or presymptomatic individuals. Real-time PCR with fluorescent hybridization is the most available, highly sensitive and specific technique for SARS-CoV-2 RNA detection in biological samples. More RT-PCR assay kits are needed for mass screening, which will help to identify infected individuals and contain the current outbreak of COVID-19 in Russia.

scholarly journals Sensitive one-step isothermal detection of pathogen-derived RNAs

2020 ◽  
Author(s):  
Chang Ha Woo ◽  
Sungho Jang ◽  
Giyoung Shin ◽  
Gyoo Yeol Jung ◽  
Jeong Wook Lee
Keyword(s):  
Diagnostic Method ◽  
High Sensitivity ◽  
T7 Rna Polymerase ◽  
Molecular Diagnostic ◽  
Enzymatic Reactions ◽  
Rna Aptamer ◽  
Rna Detection ◽  
Highly Sensitive ◽  
One Step ◽  
Sensitivity Specificity

AbstractThe recent outbreaks of Ebola, Zika, MERS, and SARS-CoV-2 (2019-nCoV) require fast, simple, and sensitive onsite nucleic acid diagnostics that can be developed rapidly to prevent the spread of diseases. We have developed a SENsitive Splint-based one-step isothermal RNA detection (SENSR) method for rapid and straightforward onsite detection of pathogen RNAs with high sensitivity and specificity. SENSR consists of two simple enzymatic reactions: a ligation reaction by SplintR ligase and subsequent transcription by T7 RNA polymerase. The resulting transcript forms an RNA aptamer that induces fluorescence. Here, we demonstrate that SENSR is an effective and highly sensitive method for the detection of the current epidemic pathogen, severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). We also show that the platform can be extended to the detection of five other pathogens. Overall, SENSR is a molecular diagnostic method that can be developed rapidly for onsite uses requiring high sensitivity, specificity, and short assaying times.

Sign in / Sign up

Export Citation Format

Share Document