scholarly journals COVID-19 Variant Detection with a High-Fidelity CRISPR-Cas12 Enzyme

2021 ◽  
Author(s):  
Clare L Fasching ◽  
Venice Servellita ◽  
Bridget McKay ◽  
Vaishnavi Nagesh ◽  
James P Broughton ◽  
...  
Keyword(s):  
Public Health ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Analysis Pipeline ◽  
Single Nucleotide ◽  
Variant Detection ◽  
Development And Validation ◽  
Data Analysis Pipeline

Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants have the potential to guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here we present the development and validation of a COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K, and N501Y mutations associated with nearly all circulating viral lineages. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all three targeted SNP mutations. We developed a data analysis pipeline for CRISPR-based SNP identification using the assay from 91 clinical samples (Ct < 30), yielding an overall SNP concordance and agreement with SARS-CoV-2 lineage classification of 100% compared to viral whole-genome sequencing. These findings highlight the potential utility of CRISPR-based mutation detection for clinical and public health diagnostics.

Simultaneous determination of Mycobacterium leprae drug resistance and single nucleotide polymorphism genotype using nested multiplex PCR with amplicon sequencing

2021 ◽  
Author(s):  
Yasuhisa Iwao ◽  
Shuichi Mori ◽  
Manabu Ato ◽  
Noboru Nakata
Keyword(s):  
Drug Resistance ◽  
Single Nucleotide Polymorphism ◽  
Multiplex Pcr ◽  
Simultaneous Determination ◽  
Mycobacterium Leprae ◽  
Amplicon Sequencing ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

Mycobacterium leprae is the predominant cause of leprosy worldwide, and its genotypes can be classified into four single nucleotide polymorphism (SNP) types and 16 subtypes. Determining M. leprae drug resistance and genotype is typically done by PCR and Sanger DNA sequencing, which require substantial effort. Here we describe a rapid method involving multiplex PCR in combination with nested amplification and next generation sequence analysis that allows simultaneous determination of M. leprae drug resistance and SNP genotype directly from clinical specimens. We used this method to analyze clinical samples from two paucibacillary, nine multibacillary, and six type-undetermined leprosy patients. Regions in folP1 , rpoB , gyrA , and gyrB that determine drug resistance and those for 84 SNP-InDels in the M. leprae genome were amplified from clinical samples and their sequences were determined. The results showed that seven samples were subtype 1A, three were 1D, and seven were 3K. Three samples of the subtype 3K had folp1 mutation. The method may allow more rapid genetic analyses of M. leprae in clinical samples.

scholarly journals Spread of TEM, VIM, SHV, and CTX-Mβ-Lactamases in Imipenem-Resistant Gram-Negative Bacilli Isolated from Egyptian Hospitals

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
El sayed Hamdy Mohammed ◽  
Ahmed Elsadek Fakhr ◽  
Hanan Mohammed El sayed ◽  
Said abd Elmohsen Al Johery ◽  
Wesam Abdel Ghani Hassanein
Keyword(s):  
Direct Sequencing ◽  
Clinical Isolates ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Therapeutic Problem

Carbapenem-resistant Gram-negative bacilli resulting fromβ-lactamases have been reported to be an important cause of nosocomial infections and are a critical therapeutic problem worldwide. This study aimed to describe the prevalence of imipenem-resistant Gram-negative bacilli isolates and detection ofblaVIM,blaTEM,blaSHV,blaCTX-M-1, andblaCTX-M-9genes in these clinical isolates in Egyptian hospitals. The isolates were collected from various clinical samples, identified by conventional methods and confirmed by API 20E. Antibiotic susceptibility testing was determined by Kirby-Bauer technique and interpreted according to CLSI. Production ofblaVIM,blaTEM,blaSHV, andblaCTX-Mgenes was done by polymerase chain reaction (PCR). Direct sequencing from PCR products was subsequently carried out to identify and confirm theseβ-lactamases genes. Out of 65 isolates, (46.1%) Escherichia coli, (26.2%) Klebsiella pneumoniae, and (10.7%) Pseudomonas aeruginosa were identified as the commonest Gram-negative bacilli. 33(50.8%) were imipenem-resistant isolates. 22 isolates (66.7%) carriedblaVIM, 24(72.7%) hadblaTEM, and 5(15%) showedblaSHV, while 12(36%), 6(18.2%), and 0(0.00%) harboredblaCTX-M-1,blaCTX-M-9, andblaCTX-M-8/25, respectively. There is a high occurrence ofβ-lactamase genes in clinical isolates and sequence analysis of amplified genes showed differences between multiple SNPs (single nucleotide polymorphism) sites in the same gene among local isolates in relation to published sequences.

scholarly journals Single-Nucleotide-Polymorphism Genotyping ofCoxiella burnetiiduring a Q Fever Outbreak in The Netherlands

2011 ◽  
Vol 77 (6) ◽  
pp. 2051-2057 ◽  
Author(s):  
Cornelis J. J. Huijsmans ◽  
Jeroen J. A. Schellekens ◽  
Peter C. Wever ◽  
Rudolf Toman ◽  
Paul H. M. Savelkoul ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
The Netherlands ◽  
Q Fever ◽  
Snp Genotyping ◽  
Cow Milk ◽  
Clinical Samples ◽  
Nucleotide Polymorphism ◽  
Animal Reservoir ◽  
Single Nucleotide ◽  
Outbreak Area

ABSTRACTCoxiella burnetiiis the etiological agent of Q fever. Currently, the Netherlands is facing the largest Q fever epidemic ever, with almost 4,000 notified human cases. Although the presence of a hypervirulent strain is hypothesized, epidemiological evidence, such as the animal reservoir(s) and genotype of theC. burnetiistrain(s) involved, is still lacking. We developed a single-nucleotide-polymorphism (SNP) genotyping assay directly applicable to clinical samples. Ten discriminatory SNPs were carefully selected and detected by real-time PCR. SNP genotyping appeared to be highly suitable for discrimination ofC. burnetiistrains and easy to perform with clinical samples. With this new method, we show that the Dutch outbreak is caused by at least 5 differentC. burnetiigenotypes. SNP typing of 14 human samples from the outbreak revealed the presence of 3 dissimilar genotypes. Two genotypes were also present in livestock at 9 farms in the outbreak area. SNP analyses of bulk milk from 5 other farms, commercial cow milk, and cow colostrum revealed 2 additional genotypes that were not detected in humans. SNP genotyping data from clinical samples clearly demonstrate that at least 5 differentC. burnetiigenotypes are involved in the Dutch outbreak.

scholarly journals Supplementing SARS-CoV-2 genomic surveillance with PCR-based variant detection for real-time actionable information, the Netherlands, June to July 2021

2021 ◽  
Vol 26 (40) ◽  
Author(s):  
Richard Molenkamp ◽  
Ewout Fanoy ◽  
Leonie Derickx ◽  
Theun de Groot ◽  
Marcel Jonges ◽  
...  
Keyword(s):  
Public Health ◽  
The Netherlands ◽  
Relative Abundance ◽  
Rapid Identification ◽  
Regional Hospital ◽  
Rt Pcr ◽  
Routine Testing ◽  
Local Public Health ◽  
Health Measures ◽  
Variant Detection

We evaluated routine testing with SARS-CoV-2 Delta variant-specific RT-PCR in regional hospital laboratories in addition to centralised national genomic surveillance in the Netherlands during June and July 2021. The increase of the Delta variant detected by RT-PCR correlated well with data from genomic surveillance and was available ca 2 weeks earlier. This rapid identification of the relative abundance and increase of SARS-CoV-2 variants of concern may have important benefits for implementation of local public health measures.

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