scholarly journals A novel CRISPR-based malaria diagnostic capable of Plasmodium detection, speciation, and drug-resistance genotyping

2020 ◽  
Author(s):  
CH Cunningham ◽  
CM Hennelly ◽  
JT Lin ◽  
R Ubalee ◽  
RM Boyce ◽  
...  
Keyword(s):  
False Negative ◽  
High Sensitivity ◽  
Disease Diagnosis ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Robust Detection ◽  
Drug Resistance Genotyping ◽  
Republic Of The Congo ◽  
Species Specific ◽  
Analytical Sensitivities

ABSTRACTCRISPR-based diagnostics are a new class of highly sensitive and specific assays with multiple applications in infectious disease diagnosis. SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing, is one such CRISPR-based diagnostic that combines recombinase polymerase pre-amplification, CRISPR-RNA base-pairing, and LwCas13a activity for nucleic acid detection. We developed SHERLOCK assays for malaria capable of detecting all Plasmodium species known to cause malaria in humans and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We validated these assays against parasite genomic DNA and achieved analytical sensitivities ranging from 2.5-18.8 parasites per reaction. We further tested these assays using a diverse panel of 123 clinical samples from the Democratic Republic of the Congo, Uganda, and Thailand and pools of Anopheles mosquitoes from Thailand. When compared to real-time PCR, the P. falciparum assay achieved 94% sensitivity and 94% specificity in clinical samples. In addition, we developed a SHERLOCK assay capable of detecting the dihydropteroate synthetase (dhps) single nucleotide variant A581G associated with P. falciparum sulfadoxine-pyrimethamine resistance. Compared to amplicon-based deep sequencing, the dhps SHERLOCK assay achieved 73% sensitivity and 100% specificity when applied to a panel of 43 clinical samples, with false-negative calls only at lower parasite densities. These novel SHERLOCK assays have potential to spawn a new generation of molecular diagnostics for malaria and demonstrate the versatility of CRISPR-based diagnostic approaches.One-sentence summaryNovel malaria SHERLOCK assays enabled robust detection, speciation, and genotyping of Plasmodium spp. in diverse samples collected in Africa and Asia.

scholarly journals RT-dPCR in Mosquito Samples for ZIKV Detection: Effects of RNA Extraction and Reverse Transcription in Target Concentration

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 827
Author(s):  
Paula Rodrigues de Almeida ◽  
Ana Karolina Antunes Eisen ◽  
Meriane Demoliner ◽  
Fernando Rosado Spilki
Keyword(s):  
Reverse Transcription ◽  
Tissue Concentration ◽  
Critical Role ◽  
Rna Extraction ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Assay Sensitivity ◽  
Robust Detection ◽  
Detection Techniques ◽  
Mosquito Pool

Zika virus (ZIKV) is an important arbovirus, responsible for recent outbreaks of Guillain Barré Syndrome and Congenital Zika Syndrome (CZS). After thousands of CZS cases, ZIKV is under constant surveillance in Brazil. Reliable and robust detection techniques are required to minimize the influence of host inhibitors from clinical samples and mosquito pool samples. Reverse transcription Digital Polymerase Chain Reaction (RT-dPCR) is a technique that allows the accurate quantification of DNA targets with high sensitivity, and it is usually less affected by inhibitors than RT-qPCR. This study aimed to assess the influence of mosquito tissue, RNA extraction and cDNA synthesis in ZIKV PCR detection. Samples containing 0, 3 and 10 mosquitoes were spiked with ZIKV MR766 and serially diluted prior to RNA extraction and RT-dPCR for ZIKV. Two reverse transcription protocols were tested. Assay sensitivity allowed the detection of 1.197 copies/µL. A higher correlation between dilution factor and target quantification was observed in 10 mosquito pool samples. The lower quantification in samples diluted without mosquitoes highlights the critical role of the reverse transcription step in RNA detection, since it could be attributed to reverse transcriptase variable performance in samples with low overall RNA concentration. The results in mosquito pools indicate that mosquito tissues do not inhibit ZIKV RT-dPCR, and the RT-dPCR technique has good sensitivity and robustness for ZIKV detection in mosquito pool samples regardless of mosquito tissue concentration.

Exploring 'best practice' for nucleic acid detection of Neisseria gonorrhoeae

Sexual Health ◽  
2008 ◽  
Vol 5 (1) ◽  
pp. 17 ◽  
Author(s):  
David M. Whiley ◽  
Suzanne M. Garland ◽  
Geoffrey Harnett ◽  
Gary Lum ◽  
David W. Smith ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Neisseria Gonorrhoeae ◽  
Best Practice ◽  
Current Knowledge ◽  
False Negative ◽  
High Sensitivity ◽  
Nucleic Acid Detection ◽  
Predictive Values ◽  
Neisseria Species ◽  
Negative Results

Nucleic acid detection tests (NADT) have considerable benefits for the detection of Neisseria gonorrhoeae (GC), including high sensitivity across a range of specimen types and use under widely differing settings and conditions. However, sexual health practitioners and others who use data generated by NADT for GC should be aware of some important limitations of these tests. False-positive results caused by cross reaction with commensal Neisseria species have been observed in many assays, and have lead to unacceptably low positive-predictive values in some patient populations. Further, false-negative results can be caused by GC sequence variation, with some gonococci lacking certain NADT target sequences. This review examines the issues associated with gonococcal NADT and considers best practice for use of these assays based on current knowledge. We emphasise the need for supplementary testing and extensive assay validation, and suggest appropriate strategies for these requirements irrespective of the setting in which they are used. Further, we highlight the need to maintain culture-based testing for certain specimen sites as well as for antimicrobial resistance surveillance.

scholarly journals Shine: To explore specific, sensitive and conserved biomarkers from massive microbial genomic data within intrapopulations.

2021 ◽  
Author(s):  
Cong Ji ◽  
Junbin Jack Shao
Keyword(s):  
False Negative ◽  
Genomic Data ◽  
Cost Effective ◽  
Pcr Primers ◽  
Detection Sensitivity ◽  
Nucleic Acid Detection ◽  
Pathogenic Microorganisms ◽  
New Strategy ◽  
Microbial Genomic ◽  
Species Specific

To improve the quality of nucleic acid detection reagents, we provided a new strategy, Shine, to explore specific, sensitive and conserved biomarkers from massive microbial genomic data within intrapopulations in order to improve detection sensitivity and accuracy. It is obvious that the more comprehensive genomic data are, the more effective the detection biomarkers. Here, we demonstrated that our method could detect undiscovered multicopy conserved species-specific or even subspecies-specific target fragments, according to several clinical projects. In particular, this approach was effective for any pathogenic microorganism even in incompletely assembled motifs. Based on our strategy, the detection device designed with quantitative PCR primers and probes for systematic and automated detection of pathogenic microorganisms in biological samples may cover all pathogenic microorganisms without limits based on genome annotation. On the website https://bioinfo.liferiver.com.cn, users may select different configuration parameters depending on the purpose of the project to realize routine clinical detection practices. Therefore, it is recommended that our strategy is suitable to identify shared universal phylogenetic markers with few false positive or false negative errors and to automate the design of minimal primers and probes to detect pathogenic communities with cost-effective predictive power.

scholarly journals CRISPR-cas13 enzymology rapidly detects SARS-CoV-2 fragments in a clinical setting

2020 ◽  
Author(s):  
Wahab A. Khan ◽  
Rachael E. Barney ◽  
Gregory J. Tsongalis
Keyword(s):  
Clinical Setting ◽  
High Sensitivity ◽  
Limited Resources ◽  
N Gene ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Viral Loads ◽  
New Thinking ◽  
And Performance ◽  
Selection Of

ABSTRACTThe well-recognized genome editing ability of the CRISPR-Cas system has triggered significant advances in CRISPR diagnostics. This has prompted an interest in developing new biosensing applications for nucleic acid detection. Recently, such applications have been engineered for detection of SARS-CoV-2. Increased demand for testing and consumables of RT-qPCR assays has led to the use of alternate testing options in some cases. Here we evaluate the accuracy and performance of a novel fluorescence based assay that received EUA authorization from the FDA for detecting SARS-CoV-2 in clinical samples. The Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology forms the basis of the Sherlock CRISPR SARS-CoV-2 kit using the CRISPR-Cas13a system. Our experimental strategy included selection of COVID-19 patient samples from previously validated RT-qPCR assays. Positive samples were selected based on a broad range of cycle thresholds. A total of 50 COVID-19 patient samples were correctly diagnosed with 100% accuracy (relative fluorescence ratios: N gene 95% CI 23.2-36.3, ORF1ab gene 95% CI 27.6-45.4). All controls, including RNase P, showed expected findings. Overall ratios were robustly distinct between positive and negative cases relative to the pre-established 5-fold change in fluorescence read output. We have evaluated the accuracy of detecting conserved targets of SARS-CoV-2 across a range of viral loads using the SHERLOCK CRISPR collateral detection reaction in a clinical setting. These findings demonstrate encouraging results, especially at a time when COVID-19 clinical diagnosis is in high demand; often with limited resources. This approach highlights new thinking in infectious disease identification and can be expanded to measure nucleic acids in other clinical isolates.

Nanomonitors: Electrical Immunoassays for Protein Biomarker Profiling

2008 ◽  
Vol 1106 ◽  
Author(s):  
Manish Bothara ◽  
Ravi K Reddy ◽  
Thomas Barrett ◽  
John Carruthers ◽  
Shalini Prasad
Keyword(s):  
Microelectrode Array ◽  
Performance Metrics ◽  
Point Of Care ◽  
High Sensitivity ◽  
Disease Diagnosis ◽  
Clinical Samples ◽  
Label Free ◽  
Early Disease ◽  
Alumina Membrane ◽  
Protein Biomarker

AbstractThe objective of this research is to develop a “point-of-care” device for early disease diagnosis through protein biomarker characterization. Here we present label-free, high sensitivity detection of proteins with the use of electrical immunoassays that we call Nanomonitors. The basis of the detection principle lies in the formation of an electrical double layer and its perturbations caused by proteins trapped in a nanoporous alumina membrane over a microelectrode array platform. High sensitivity and rapid detection of two inflammatory biomarkers, C-reactive protein (CRP) and Myeloperoxidase (MPO) in pure and clinical samples through label-free electrical detection were achieved. The performance metrics achieved by this device makes it suitable as a “lab-on-a-chip” device for protein biomarker profiling and hence early disease diagnosis.

Improvement and Application of qPCR (Real-Time Quantitative Polymerase Chain Reaction) Data Processing Method for Home-Made Integrated Nucleic Acid Detection System

2020 ◽  
Vol 20 (12) ◽  
pp. 7369-7375
Author(s):  
Yile Fang ◽  
Pei Liao ◽  
Zhu Chen ◽  
Hui Chen ◽  
Yanqi Wu ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Data Processing ◽  
Real Time ◽  
Detection System ◽  
Disease Diagnosis ◽  
Processing Algorithm ◽  
Clinical Samples ◽  
Nucleic Acid Detection ◽  
Qpcr Data ◽  
Data Processing Algorithm

Because it has many advantages such as rapidity and accuracy, nucleic acid detection is applied to infectious disease diagnosis more and more. An automatic integrated nucleic acid detection system based on real-time PCR is developed by our research group to conduct point-of-care testing of infectious pathogens. The home-made detection system collects fluorescence data in each PCR cycle through an integrated dual-channel fluorescence detection module and then real-time fluorescence curves are drawn by the software, which can tell the results of the diagnostics after some processing and analysis. However, owing to the disturbance of the environment or the imperfect of nucleic acid extraction before PCR, the fluorescence curves sometimes may contain several abnormal points. For the purpose of enhancing its ability to deal with these iffy curves and improve the accuracy of the testing results, in this study, the SDM-based qPCR data processing algorithm was studied and 11 groups of qPCR data that have different flaws from the clinical samples detected by this system were chosen to prove the practicability of the method. In comparison with the conventional threshold-based method, the Cq values calculated by the SDM-based method were more close to the actual values, meaning it can overcome the shortcomings of the conventional methods such as being unable to accommodate noise and being unable to avoiding abnormal data. With the improvement of this data processing algorithm, the stability of our system and the reliability and accuracy of the results are greatly improved.

DETECTING MYCOBACTERIUM TUBERCULOSIS RAPIDLY AND MYCOBACTERIUM TUBERCULOSIS STRAINS WITHOUT IS6110 SEQUENCE BY PCR ASSAY

2012 ◽  
pp. 15-19
Author(s):  
Thi Chau Anh Nguyen ◽  
Hoang Bach Nguyen ◽  
Hai Duong Huynh ◽  
Nu Xuan Thanh Le ◽  
Xuan Cuong Le ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rdna ◽  
False Negative ◽  
Clinical Samples ◽  
Tuberculosis Complex ◽  
Pcr Assay ◽  
Complex Objects ◽  
Method Performance ◽  
Gene Coding ◽  
16S Rdna Pcr

Background: The Nested IS6110 PCR is used for detecting tuberculosis, however IS6110 sequence is not present in the genome of all strains of M.tuberculosis, the result may be false negative. The gene coding 16S ribosome always contains a short sequence specific to M. tuberculosis complex. Objects: Performance of the 16S Real-time PCR to detect M. tuberculosis and combining to the nested IS6110 PCR to determine the rate of Mtb strains without IS6110 from clinical samples. Materials and method: Performance of 16S rDNA PCR by commercial kit of Viet A Inc. for all 480 samples, the samples which were positive with the 16S rDNA PCR were retested in IS6110 PCR assay by in-house kit. Results: The Realtime 16S rDNA PCR detected 258 cases (53.8%) of tuberculosis. There were 3 (1.2 %) M. tuberculosis strains which do not harbor IS6110 sequence in genome. Conclusion: The IS6110 nested PCR can be applied more widely than the 16S rDNA realtime PCR. In case of using IS6110 PCR assay, results may show a low proportion of false negative. Combining 16S rDNA PCR with the IS6110 based PCR allowed detection of deletion of IS6110 sequence in M. tuberculosis isolates.

scholarly journals Essential properties and pitfalls of colorimetric Reverse Transcription Loop-mediated Isothermal Amplification as a point-of-care test for SARS-CoV-2 diagnosis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Bruna de Oliveira Coelho ◽  
Heloisa Bruna Soligo Sanchuki ◽  
Dalila Luciola Zanette ◽  
Jeanine Marie Nardin ◽  
Hugo Manuel Paz Morales ◽  
...  
Keyword(s):  
Viral Load ◽  
Internal Control ◽  
Reverse Transcription ◽  
Color Change ◽  
Point Of Care ◽  
Colorimetric Detection ◽  
False Negative ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Loop Mediated Isothermal Amplification

Abstract Background SARS-CoV-2 Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) colorimetric detection is a sensitive and specific point-of-care molecular biology technique used to detect the virus in only 30 min. In this manuscript we have described a few nuances of the technique still not properly described in the literature: the presence of three colors clusters; the correlation of the viral load with the color change; and the importance of using an internal control to avoid false-negative results. Methods To achieve these findings, we performed colorimetric RT-LAMP assays of 466 SARS-CoV-2 RT-qPCR validated clinical samples, with color quantification measured at 434 nm and 560 nm. Results First we determinate a sensitivity of 93.8% and specificity of 90.4%. In addition to the pink (negative) and yellow (positive) produced colors, we report for the first time the presence of an orange color cluster that may lead to wrong diagnosis. We also demonstrated using RT-qPCR and RT-LAMP that low viral loads are related to Ct values > 30, resulting in orange colors. We also demonstrated that the diagnosis of COVID-19 by colorimetric RT-LAMP is efficient until the fifth symptoms day when the viral load is still relatively high. Conclusion This study reports properties and indications for colorimetric RT-LAMP as point-of-care for SARS-CoV-2 diagnostic, reducing false results, interpretations and optimizing molecular diagnostics tests application.

scholarly journals A Review of Murine Cytomegalovirus as a Model for Human Cytomegalovirus Disease—Do Mice Lie?

2020 ◽  
Vol 22 (1) ◽  
pp. 214
Author(s):  
Michelle A. Fisher ◽  
Megan L. Lloyd
Keyword(s):  
Human Cytomegalovirus ◽  
Murine Cytomegalovirus ◽  
Clinical Samples ◽  
Mouse Strains ◽  
Cytomegalovirus Disease ◽  
Wild Mice ◽  
Congenital Cytomegalovirus ◽  
Non Invasive ◽  
Species Specific ◽  
Disseminated Disease

Since murine cytomegalovirus (MCMV) was first described in 1954, it has been used to model human cytomegalovirus (HCMV) diseases. MCMV is a natural pathogen of mice that is present in wild mice populations and has been associated with diseases such as myocarditis. The species-specific nature of HCMV restricts most research to cell culture-based studies or to the investigation of non-invasive clinical samples, which may not be ideal for the study of disseminated disease. Initial MCMV research used a salivary gland-propagated virus administered via different routes of inoculation into a variety of mouse strains. This revealed that the genetic background of the laboratory mice affected the severity of disease and altered the extent of subsequent pathology. The advent of genetically modified mice and viruses has allowed new aspects of disease to be modeled and the opportunistic nature of HCMV infection to be confirmed. This review describes the different ways that MCMV has been used to model HCMV diseases and explores the continuing difficulty faced by researchers attempting to model HCMV congenital cytomegalovirus disease using the mouse model.

scholarly journals Comparison between Mononucleotide and Dinucleotide Marker Panels in Gastric Cancer with Loss of hMLH1 or hMSH2 Expression

2017 ◽  
Vol 32 (3) ◽  
pp. 352-356 ◽  
Author(s):  
Jeong Goo Kim ◽  
Soyoung Shin ◽  
Joonhong Park
Keyword(s):  
Gastric Cancer ◽  
High Sensitivity ◽  
Size Change ◽  
Marker Panel ◽  
Dna Mismatch ◽  
Repair Deficiency ◽  
Msi Analysis ◽  
Analytical Sensitivities ◽  
Dna Mismatch Repair Deficiency

Background DNA mismatch repair deficiency is an important molecular mechanism of genetic instability in gastric cancer, and a high instability at microsatellites is associated with favorable prognosis. We compared mononucleotide and dinucleotide microsatellite instability (MSI) marker panels in 56 paired gastric tumor and normal samples. Methods The mononucleotide marker panel (mono panel) consisted of 8 markers: BAT25, BAT26, BAT40, BAT-RII, NR21, NR22, NR24 and NR27. The dinucleotide marker panel (di panel) contained D2S123, D5S346, D17S250, D17S261, D17S520, D18S34 and D18S58. The NCI panel was used as reference panel. Results Among 13 gastric tumors showing no hMLH1 or hMSH2 expression, 8 MSI-H (high) and 5 MSI-L (low) were identified. The analytical sensitivities of the NCI, mono and di panels to detect unstable MSI were 61.5% (8/13), 76.9% (10/13) and 84.6% (11/13), respectively. The size change of allele shift was statistically greater in the mono panel than in the di panel (p = 0.02 by Mann-Whitney U-test). The BAT40 (69.2%, 9/13) and D18S34 (76.9%, 10/13) markers showed high sensitivity for determination of MSI status. Conclusions To improve the detection rate of MSI in gastric cancer with loss of hMLH1 or hMSH2 expression, the kind of MSI marker may need to be considered more, instead of the repetitive type of marker. Thus, an MSI panel designed with a combination of both BAT40 and D18S34 is suggested for providing more accurate and sensitive MSI analysis in gastric cancer.

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