Development of a multiplex taqMan real-time PCR assay for typing of Mycoplasma pneumoniae based on type-specific indels identified through whole genome sequencing

2017 ◽  
Vol 87 (3) ◽  
pp. 203-206 ◽  
Author(s):  
Bernard J. Wolff ◽  
Alvaro J. Benitez ◽  
Heta P. Desai ◽  
Shatavia S. Morrison ◽  
Maureen H. Diaz ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Mycoplasma Pneumoniae ◽  
Real Time Pcr ◽  
Whole Genome ◽  
Pcr Assay

scholarly journals Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

2020 ◽  
Author(s):  
AG Stewart ◽  
EP Price ◽  
K Schabacker ◽  
M Birikmen ◽  
PNA Harris ◽  
...  
Keyword(s):  
Intensive Care Unit ◽  
Intensive Care ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Real Time Pcr ◽  
Generation Cephalosporin ◽  
Whole Genome ◽  
Third Generation ◽  
Sensitivity Testing

AbstractThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 258 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using a rapid (~40 min) pentaplex real-time PCR assay targeting the most common extended spectrum β-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). Additionally, AmpC CMY β-lactamase prevalence was examined using a singleplex PCR. A subset of isolates, including all 3GC-R isolates obtained from the intensive care unit, were subjected to whole-genome sequencing (WGS) to assess transmission dynamics, the presence of unidentified resistance determinants, and genotyping accuracy. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance was identified in 97% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, the majority (98.3%) of isolates harbored at least one β-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. WGS of β-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. No evidence of transmission among intensive care unit patients was identified. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

scholarly journals Real-Time PCR Assay for Differentiation of Typhoidal and Nontyphoidal Salmonella

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Satheesh Nair ◽  
Vineet Patel ◽  
Tadgh Hickey ◽  
Clare Maguire ◽  
David R. Greig ◽  
...  
Keyword(s):  
Public Health ◽  
Risk Management ◽  
Real Time ◽  
Genome Sequencing ◽  
Real Time Pcr ◽  
Enteric Fever ◽  
Rapid Identification ◽  
Whole Genome ◽  
Pcr Assay ◽  
Health Measures

ABSTRACT Rapid and accurate differentiation of Salmonella spp. causing enteric fever from nontyphoidal Salmonella is essential for clinical management of cases, laboratory risk management, and implementation of public health measures. Current methods used for confirmation of identification, including biochemistry and serotyping as well as whole-genome sequencing analyses, take several days. Here we report the development and evaluation of a real-time PCR assay that can be performed directly on crude DNA extracts from bacterial colonies for the rapid identification of typhoidal and nontyphoidal Salmonella.

scholarly journals The 2017 Dengue virus 1 outbreak in northern Vietnam was caused by a locally circulating virus group

2022 ◽  
Vol 50 (1) ◽  
Author(s):  
Taichiro Takemura ◽  
Co Thach Nguyen ◽  
Ha Chau Pham ◽  
Thuy Trang Nguyen ◽  
Vu Mai Phuong Hoang ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Dengue Fever ◽  
Genome Sequencing ◽  
Real Time Pcr ◽  
Insect Vector ◽  
Whole Genome ◽  
Sequencing Analysis ◽  
Northern Vietnam

Abstract Background Dengue virus (DENV) is a member of insect vector-borne viruses, and it causes dengue fever. Southeast Asia is the epi-center of dengue fever in the world. The characterization of the virus is essential to identify the transmission and evolution of DENV. Objectives In 2017, there was an outbreak of Dengue virus type 1 (DENV1) in northern Vietnam and the neighboring countries. To identify the genetic character of the outbreak virus in the area, we conducted whole-genome sequencing analysis on the samples positive for the DENV1 along with real-time PCR. Study design In total, 1026 blood samples were collected from patients with suspected dengue fever in Ha Nam and Hai Duong province, nearby areas of the capital of Vietnam. After screening by real-time PCR, 40 of DENV1 positive samples were subjected to whole-genome sequencing, and 28 complete coding sequences were obtained. Results All 28 sequences were genotype I of DENV1, which is dominant in the southeast and East Asian countries. The phylogenetic analysis of the E region showed that they fell into a single cluster with the reported sequences from Vietnam between 2009 and 2016, in which the isolates from other countries are very rare. Our results suggested that the 2017 outbreak in the area was caused by locally circulating viruses.

scholarly journals Detection of Salmonella enterica subsp. enterica Serovar Cubana from Naturally Contaminated Chick Feed

2017 ◽  
Vol 80 (11) ◽  
pp. 1815-1820 ◽  
Author(s):  
Faiza Benahmed ◽  
Hua Wang ◽  
Junia Jean-Gilles Beaubrun ◽  
Gopal R. Gopinath ◽  
Chorng-Ming Cheng ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Culture Method ◽  
Whole Genome ◽  
Molecular Serotyping ◽  
Peptone Water ◽  
Pure Cultures

ABSTRACTBecause some significant outbreaks of human salmonellosis have been traced to contaminated animal feed, the rapid and efficient detection of Salmonella in feed is essential. However, the current U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) culture method that uses lactose broth as a preenrichment medium has not reliably supported the results of real-time PCR assays for certain foods. We evaluated the BAM culture method and a quantitative real-time PCR (qPCR) assay using two preenrichment media, modified buffered peptone water and lactose broth, to detect Salmonella enterica subsp. enterica serovar Cubana in naturally contaminated chick feed. After 24 h of incubation, the qPCR method was as sensitive as the culture method when modified buffered peptone water was used as the preenrichment medium but less sensitive than culture when lactose broth was used. After 48 h of incubation, detection of Salmonella Cubana by qPCR and by culture in either preenrichment medium was equivalent. We also compared the performance of the traditional serotyping method, which uses pure cultures of Salmonella grown on blood agar, to two molecular serotyping methods. The serotyping method based on whole genome sequencing also requires pure cultures, but the PCR-based molecular serotyping method can be done directly with the enriched culture medium. The PCR-based molecular serotyping method provided simple and rapid detection and identification of Salmonella Cubana. However, whole genome sequencing allows accurate identification of many Salmonella serotypes and highlights variations in the genomes, even in tight genomic clusters. We also compared the genome of the chick feed isolate with 58 Salmonella Cubana strains in GenBank and found that the chick feed isolate was very closely related to an isolate from a foodborne outbreak involving alfalfa sprouts.

scholarly journals High-precision and cost-efficient sequencing for real-time COVID-19 surveillance

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sung Yong Park ◽  
Gina Faraci ◽  
Pamela M. Ward ◽  
Jane F. Emerson ◽  
Ha Youn Lee
Keyword(s):  
Los Angeles ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
High Precision ◽  
High Throughput Sequencing ◽  
Whole Genome ◽  
Sequencing Data ◽  
Public Health Response ◽  
Cost Efficient

AbstractCOVID-19 global cases have climbed to more than 33 million, with over a million total deaths, as of September, 2020. Real-time massive SARS-CoV-2 whole genome sequencing is key to tracking chains of transmission and estimating the origin of disease outbreaks. Yet no methods have simultaneously achieved high precision, simple workflow, and low cost. We developed a high-precision, cost-efficient SARS-CoV-2 whole genome sequencing platform for COVID-19 genomic surveillance, CorvGenSurv (Coronavirus Genomic Surveillance). CorvGenSurv directly amplified viral RNA from COVID-19 patients’ Nasopharyngeal/Oropharyngeal (NP/OP) swab specimens and sequenced the SARS-CoV-2 whole genome in three segments by long-read, high-throughput sequencing. Sequencing of the whole genome in three segments significantly reduced sequencing data waste, thereby preventing dropouts in genome coverage. We validated the precision of our pipeline by both control genomic RNA sequencing and Sanger sequencing. We produced near full-length whole genome sequences from individuals who were COVID-19 test positive during April to June 2020 in Los Angeles County, California, USA. These sequences were highly diverse in the G clade with nine novel amino acid mutations including NSP12-M755I and ORF8-V117F. With its readily adaptable design, CorvGenSurv grants wide access to genomic surveillance, permitting immediate public health response to sudden threats.

scholarly journals Genome Sequence ofListeria monocytogenesStrain HPB2088 (Serotype 1/2a), an Environmental Isolate Collected in Canada in 1994

2016 ◽  
Vol 4 (4) ◽  
Author(s):  
Arthur W. Pightling ◽  
Hugh Rand ◽  
Errol Strain ◽  
Franco Pagotto
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Genome Sequence ◽  
Foodborne Pathogen ◽  
Environmental Isolate ◽  
Severe Illness ◽  
Whole Genome ◽  
Serotype 1

Listeria monocytogenesis a foodborne pathogen that causes severe illness. Thus, ongoing efforts at real-time whole-genome sequencing are of utmost importance. However, it is also important that retrospective analyses that place these data into context be performed. Here, we present the genome sequence of strain HPB2088, which was collected in 1994.

scholarly journals Real-Time Whole-Genome Sequencing for Surveillance ofListeria monocytogenes, France

2017 ◽  
Vol 23 (9) ◽  
pp. 1462-1470 ◽  
Author(s):  
Alexandra Moura ◽  
Mathieu Tourdjman ◽  
Alexandre Leclercq ◽  
Estelle Hamelin ◽  
Edith Laurent ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Oflisteria Monocytogenes

scholarly journals Real time, field-deployable whole genome sequencing of malaria parasites using nanopore technology

2020 ◽  
Author(s):  
Zahra Razook ◽  
Somya Mehra ◽  
Brittany Gilchrist ◽  
Digjaya Utama ◽  
Dulcie Lautu-Gumal ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Real Time ◽  
Genome Sequencing ◽  
Error Rates ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Virulence Determinants ◽  
Malaria Parasites ◽  
Additional Information ◽  
Natural Isolates

ABSTRACTMalaria parasite genomes have been generated predominantly using short read sequencing technology which can be slow, requires advanced laboratory training and does not adequately interrogate complex genomic regions that harbour important malaria virulence determinants. The portable Oxford Nanopore Technologies MinION platform generates long reads in real time and may overcome these limitations. We present compelling evidence that Nanopore sequencing delivers valuable additional information for malaria parasites with comparable data fidelity for single nucleotide variant (SNV) calls, compared to standard Illumina whole genome sequencing. We demonstrate this through sequencing of pure Plasmodium falciparum DNA, mock infections and natural isolates. Nanopore has low error rates for haploid SNV genotyping and identifies structural variants (SVs) not detected with short reads. Nanopore genomes are directly comparable to publically available genomes and produce high quality end to end chromosome assemblies. Nanopore sequencing will expedite genomic surveillance of malaria and provide new insights into parasite genome biology.

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