scholarly journals Cas12a and Lateral Flow Strip-Based Test for Rapid and Ultrasensitive Detection of Spinal Muscular Atrophy

Biosensors ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 154
Author(s):  
Chunhua Zhang ◽  
Zhuo Li ◽  
Miaomiao Chen ◽  
Zhiqing Hu ◽  
Lingqian Wu ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Muscular Atrophy ◽  
Turnaround Time ◽  
Lateral Flow ◽  
Practical Significance ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Great Promise ◽  
Minimum Detectable Concentration ◽  
Lateral Flow Strip

Spinal muscular atrophy (SMA) is characterized by severe lethality and irreversible progression. Early diagnosis of SMA is of more practical significance with the emergence of effective therapy. However, existing techniques to identify SMA patients rely on cumbersome instruments, hindering their accessibility and application. An SMA-Cas12a-strip assay was developed with the integration of Cas12a-based nucleic acid detection, isothermal amplification, and lateral flow strip. The analytical performance of the assay was assessed with clinical samples. To explore its extensible utility, various specimens were tested. Validated with 168 clinical samples, the sensitivity and specificity of the SMA-Cas12a-strip assay were both 100%. The minimum detectable concentration of genomic DNA containing the target gene achieved 526 aM. The assay was compatible with specimens from several sources, and the turnaround time could be within 1.5 h. We developed a simple, cost-effective, and highly sensitive and specific assay to detect SMA patients. With little and field-portable equipment, the assay holds great promise in the detection of SMA patients, particularly in low-resource regions.

scholarly journals Development and Performance verification of colloidal gold labeled SARS-CoV-2 antigen detection method for routine popular screening of COVID-19 with clinical samples in Poland and China

2021 ◽  
Author(s):  
Chuanxiang Guo ◽  
Li Yao ◽  
Fengling Chen ◽  
Chao Zhang ◽  
Wei Chen
Keyword(s):  
Predictive Value ◽  
Colloidal Gold ◽  
Detection Method ◽  
Age Distribution ◽  
Antigen Detection ◽  
Lateral Flow ◽  
Rapid Screening ◽  
Clinical Samples ◽  
Lateral Flow Strip ◽  
And Performance

In this research, we have constructed and optimized the colloidal gold labeled lateral flow strip (LFS) for rapid detection of antigen of SARS-CoV-2 and rapid screening of COVID-19. Based on the constructed and optimized colloidal gold lateral flow strip, the parameters of the LFS have been well evaluated with the clinical samples in the professional labs. The screening performance have also been evaluated from the aspects including the CT values, age distribution and onset of symptoms. Finally, based on the detection results of 420 clinical samples, the LFS can achieve the screening of COVID-19 with the positive percentage agreement (PPA, sensitivity), negative percent agreement (NPA, specificity), the positive predictive value (PPV) and the negative predictive value (NPV) of 96.8%, 100%, 100% and 96.6%, respectively, indicating the powerful potential for practical screening applications in pandemic control. Of great significance, this developed SARS-CoV-2 antigen detection method has also been successfully utilized for screening of delta-variant of SARS-CoV-2.

scholarly journals Antisense Oligonucleotide Therapies for Neurodegenerative Diseases

2019 ◽  
Vol 42 (1) ◽  
pp. 385-406 ◽  
Author(s):  
C. Frank Bennett ◽  
Adrian R. Krainer ◽  
Don W. Cleveland
Keyword(s):  
Neurodegenerative Diseases ◽  
Spinal Muscular Atrophy ◽  
Antisense Oligonucleotide ◽  
Antisense Oligonucleotides ◽  
Muscular Atrophy ◽  
Neurological Diseases ◽  
Great Promise ◽  
Disease Symptoms ◽  
Therapeutic Platform ◽  
Lateral Sclerosis

Antisense oligonucleotides represent a novel therapeutic platform for the discovery of medicines that have the potential to treat most neurodegenerative diseases. Antisense drugs are currently in development for the treatment of amyotrophic lateral sclerosis, Huntington's disease, and Alzheimer's disease, and multiple research programs are underway for additional neurodegenerative diseases. One antisense drug, nusinersen, has been approved for the treatment of spinal muscular atrophy. Importantly, nusinersen improves disease symptoms when administered to symptomatic patients rather than just slowing the progression of the disease. In addition to the benefit to spinal muscular atrophy patients, there are discoveries from nusinersen that can be applied to other neurological diseases, including method of delivery, doses, tolerability of intrathecally delivered antisense drugs, and the biodistribution of intrathecal dosed antisense drugs. Based in part on the early success of nusinersen, antisense drugs hold great promise as a therapeutic platform for the treatment of neurological diseases.

scholarly journals One-Tube SARS-CoV-2 Detection Platform Based on RT-RPA and CRISPR/Cas12a

2020 ◽  
Author(s):  
Yangyang Sun ◽  
Lei Yu ◽  
Chengxi Liu ◽  
Wei Chen ◽  
Dechang Li ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Limit Of Detection ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
N Gene ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Detection Process ◽  
Human Coronaviruses ◽  
Negative Controls

Abstract Background: COVID-19 has spread rapidly around the world, affecting almost every person. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. Methods: We designed RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) primers of RdRp gene and N gene according to the SARS-CoV-2 gene sequence. We optimized the components in the reaction so that the detection process could be carried out in one tube. The specificity was demonstrated through detecting nucleic acid samples from seven human coronaviruses. Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards diluted by different gradients were used to demonstrate the limit of detection. Furthermore, we have developed a lateral flow assay based on OR-DETECTR for the detection of COVID-19. Results: We have developed a o ne-tube detection platform based on R T- R PA and DNA Endonuclease-Targeted CRISPR Trans Reporter ( DETECTR ) technology, termed OR-DETECTR, to detect SARS-CoV-2. The detection process is completed in one tube, and the time is 50min. The method can specifically detect SARS-CoV-2 from seven human coronaviruses with a low detection limit of 2.5 copies/µl input. Results from six SARS-CoV-2 patient samples, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. Furthermore, we have developed a lateral flow assay based on OR-DETECTR for the detection of COVID-19. Conclusions: OR-DETECTR detection platform is rapid, accurate, tube closed, easy-to-operate, and free of large instruments for COVID-19 detection.

scholarly journals Cas12a-based on-site and rapid nucleic acid detection of African swine fever

2019 ◽  
Author(s):  
Jing Bai ◽  
Haosi Lin ◽  
Haojian Li ◽  
Yang Zhou ◽  
Junshan Liu ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Low Cost ◽  
African Swine Fever ◽  
Lateral Flow ◽  
Ease Of Use ◽  
Nucleic Acid Detection ◽  
Lateral Flow Strip ◽  
Trade Offs ◽  
Laboratory Facilities ◽  
Sensitivity Specificity

AbstractThe mortality rate of hemorrhagic African swine fever (ASF), which targets domestic pigs and is caused by African swine fever virus (ASFV), can reach 100%. ASF has been reported in 25 Chinese provinces since August 2018. There is no effective treatment or vaccine for it and the present molecular diagnosis technologies have trade-offs in sensitivity, specificity, cost and speed, and none of them cater perfectly to ASF control. Thus, a technology that overcomes the need for laboratory facilities, is relatively low cost, and rapidly and sensitively detects ASFV would be highly valuable. Here, we describe an RAA-Cas12a-based system that combines recombinase-aided amplification (RAA) and CRISPR/Cas12a for ASFV detection. The fluorescence intensity readout of this system detected ASFV p72 gene levels as low as 10 aM. For on-site ASFV detection, lateral-flow strip readout was introduced for the first time in the RAA-Cas12a based system (named CORDS, Cas12a-based On-site and Rapid Detection System). We used CORDS to detect target DNA highly specifically using the lateral-flow strip readout. CORDS could identify the p72 gene at femtomolar sensitivity in an hour at 37°C, and only requires an incubator. For ease of use, the regents of CORDS was lyophilized to three tubes and remained the same sensitivity when stored at 4 °C for at least 7 days. Thus, CORDS provides a rapid, sensitive and easily operable method for ASFV on-site detection. Lyophilized CORDS can withstand long-term transportation and storage, and is ready for field applications.

scholarly journals One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

2021 ◽  
Vol 12 ◽  
Author(s):  
Jia Xie ◽  
Xiaohan Yang ◽  
Lei Duan ◽  
Keyi Chen ◽  
Pan Liu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Effective Vaccine ◽  
Lateral Flow Strip ◽  
Coxsackievirus A6 ◽  
One Step

Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.

scholarly journals One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yangyang Sun ◽  
Lei Yu ◽  
Chengxi Liu ◽  
Shanting Ye ◽  
Wei Chen ◽  
...  
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Influenza A ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Detection Process ◽  
Influenza A H1n1 ◽  
Human Coronaviruses

Abstract Background COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. Methods We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19. Results The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input. Conclusions The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

scholarly journals Lateral Flow Aptasensor for Simultaneous Detection of Platelet-Derived Growth Factor-BB (PDGF-BB) and Thrombin

Molecules ◽  
2019 ◽  
Vol 24 (4) ◽  
pp. 756 ◽  
Author(s):  
Guodong Liu ◽  
Anant Gurung ◽  
Wanwei Qiu
Keyword(s):  
Growth Factor ◽  
Point Of Care ◽  
Low Cost ◽  
Simultaneous Detection ◽  
Lateral Flow ◽  
Platelet Derived Growth Factor ◽  
Great Promise ◽  
Serum Samples ◽  
Lateral Flow Strip ◽  
Assay Time

Here we report a lateral flow aptasensor (LFA) for the simultaneous detection of platelet-derived growth factor-BB (PDGF-BB) and thrombin. Two pairs of aptamers, which are specific against PDGF-BB and thrombin, respectively, were used to prepare the LFA. Thiolated aptamers were immobilized on a gold nanoparticle (AuNP) surface and biotinylated aptamers were immobilized on the test zones of an LFA nitrocellulose membrane. The assay involved the capture of PDGF-BB and thrombin simultaneously in sandwich-type formats between the capture aptamers on the test zones of LFA and AuNP-labeled detection aptamers. AuNPs were thus captured on the test zones of the LFA and gave red bands to enable the visual detection of target proteins. Quantitative results were obtained by reading the test band intensities with a portable strip reader. By combining the highly specific molecular recognition properties of aptamers with the unique properties of lateral flow assay (low-cost, short assay time and a user-friendly format), the optimized aptasensor was capable of simultaneously detecting 1.0 nM of PDGF-BB and 1.5 nM of thrombin in association with a 10-min assay time. The biosensor was also successfully applied to detect PDGF-BB and thrombin in spiked human serum samples. The LFA shows great promise for the development of aptamer-based lateral flow strip biosensors for point-of-care or for the in-field detection of disease-related protein biomarkers.

scholarly journals Development and validation of a 4-color multiplexing spinal muscular atrophy (SMA) genotyping assay on a novel integrated digital PCR instrument

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Lingxia Jiang ◽  
Robert Lin ◽  
Steve Gallagher ◽  
Andrew Zayac ◽  
Matthew E. R. Butchbach ◽  
...  
Keyword(s):  
Spinal Muscular Atrophy ◽  
Copy Number ◽  
Muscular Atrophy ◽  
Digital Pcr ◽  
Copy Number Variations ◽  
Higher Order ◽  
Clinical Samples ◽  
Internal Reference ◽  
Genotyping Assay ◽  
Copy Numbers

AbstractDigital PCR (dPCR) technology has been proven to be highly sensitive and accurate in detecting copy number variations (CNV). However, a higher-order multiplexing dPCR assay for measuring SMN1 and SMN2 copy numbers in spinal muscular atrophy (SMA) samples has not been reported. Described here is a rapid multiplex SMA dPCR genotyping assay run on a fully integrated dPCR instrument with five optical channels. The hydrolysis probe-based multiplex dPCR assay quantifies SMN1, SMN2, and the total SMN (SMN1 + SMN2) while using RPPH1 gene as an internal reference control. The quadruplex assay was evaluated with characterized control DNA samples and validated with 15 blinded clinical samples from a previously published study. SMN1 and SMN2 copy numbers were completely concordant with previous results for both the control and blinded samples. The dPCR-based SMA copy number determination was accomplished in 90 min with a walk-away workflow identical to real-time quantitative PCR (qPCR). In summary, presented here is a simple higher-order multiplexing solution on a novel digital PCR platform to meet the growing demand for SMA genotyping and prognostics.

scholarly journals One-tube SARS-CoV-2 detection platform based on RT-RPA and CRISPR/Cas12a

2021 ◽  
Author(s):  
Yangyang Sun ◽  
Lei Yu ◽  
Chengxi Liu ◽  
Shanting Ye ◽  
Wei Chen ◽  
...  
Keyword(s):  
Detection Limit ◽  
Nucleic Acid ◽  
Influenza A ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Detection Process ◽  
Influenza A H1n1 ◽  
Human Coronaviruses

Abstract Background: COVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component. Methods: We tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.Results: The OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.Conclusions: The OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

Sign in / Sign up

Export Citation Format

Share Document