scholarly journals Comparison of the Multiple Platforms to Identify Various Aeromonas Species

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaoli Du ◽  
Mengyu Wang ◽  
Haijian Zhou ◽  
Zhenpeng Li ◽  
Jialiang Xu ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Whole Genome Sequencing ◽  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Pcr Amplification ◽  
Identification Accuracy ◽  
Aeromonas Species ◽  
Whole Genome ◽  
Mass Spectrometry Identification ◽  
Mass Spectrometric Identification

We compared several identification methods for Aeromonas genus members, including traditional biochemical testing, multiplex-PCR amplification, mass spectrometry identification, whole-genome sequencing, multilocus phylogenetic analysis (MLPA), and rpoD, gyrA, and rpoD-gyrA gene sequencing. Isolates (n = 62) belonging to the Aeromonas genus, which were came from the bacterial bank in the laboratory, were used to assess the identification accuracy of the different methods. Whole-genome sequencing showed that the Aeromonas spp. isolates comprised A. caviae (n = 21), A. veronii (n = 18), A. dhakensis (n = 8), A. hydrophila (n = 7), A. jandaei (n = 5), A. enteropelogenes (n = 2), and A. media (n = 1). Using the whole-genome sequencing results as the standard, the consistency of the other methods was compared with them. The results were 46.77% (29/62) for biochemical identification, 83.87% (52/62) for mass spectrometric identification, 67.74% (42/62) for multiplex-PCR, 100% (62/62) for MLPA typing, 72.58% for gyrA, and 59.68% for rpoD and gyrA-rpoD. MLPA was the most consistent, followed by mass spectrometry. Therefore, in the public health laboratory, both MLPA and whole-genome sequencing methods can be used to identify various Aeromonas species. However, rapid and relatively accurate mass spectrometry is recommended for clinical lab.

scholarly journals Determination ofElizabethkingiaDiversity by MALDI-TOF Mass Spectrometry and Whole-Genome Sequencing

2017 ◽  
Vol 23 (2) ◽  
pp. 320-323 ◽  
Author(s):  
Helle Brander Eriksen ◽  
Heidi Gumpert ◽  
Cecilie Haase Faurholt ◽  
Henrik Westh
Keyword(s):  
Mass Spectrometry ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Maldi Tof Mass Spectrometry ◽  
Maldi Tof

scholarly journals A Case of IMP-4-, OXA-421-, OXA-96-, and CARB-2-Producing Acinetobacter pittii Sequence Type 119 in Australia

2014 ◽  
Vol 53 (2) ◽  
pp. 727-730 ◽  
Author(s):  
Witchuda Kamolvit ◽  
Petra Derrington ◽  
David L. Paterson ◽  
Hanna E. Sidjabat
Keyword(s):  
Whole Genome Sequencing ◽  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Draft Genome ◽  
Sequence Type ◽  
Species Level ◽  
Whole Genome ◽  
Patient Identification ◽  
Content Type ◽  
Acinetobacter Pittii

An IMP-4-producingAcinetobacterpittiistrain coproducing oxacillinases was isolated from a leg wound of a 67-year-old female patient. Identification to the species level byrpoBandgyrBsequencing and multiplex-PCR-based analysis revealed that the isolate wasA. pittii. Whole-genome sequencing of thisA. pittiiisolate determined the presence ofblaOXA-96,blaCARB-2, and a novelblaOXA-421gene. The position of this novelblaOXA-421gene was similar to that ofblaOXA-51inA. baumannii, downstream of the phosphinothricinN-acetyltransferase gene and upstream offxsAin the chromosome. ThisA. pittiiisolate was found to belong to sequence type 119 (ST119). Here, we report the first isolation of IMP-4-producingA. pittiiST119 with a novelblaOXA-421gene from a patient in Australia and characterize its draft genome.

scholarly journals Serotyping of sub-Saharan Africa Salmonella Strains Isolated from Different Sources Using Multiplex PCR and Capillary Electrophoresis Analysis and whole Genome Sequencing

2020 ◽  
Author(s):  
Assèta Kagambèga ◽  
Lari M. Hiott ◽  
David S. Boyle ◽  
Elizabeth A. McMillan ◽  
Kaisa Haukka ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Whole Genome Sequencing ◽  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Sub Saharan Africa ◽  
Whole Genome ◽  
Sub Saharan ◽  
Different Sources

Abstract The authors have withdrawn this preprint due to author disagreement.

Optimization of multiplex PCR composition to screen for SARS-CoV-2 variants of concern

2021 ◽  
Vol 4 (2) ◽  
pp. 58
Author(s):  
Maya Savira ◽  
Enikarmila Asni ◽  
Rahmat Azhari Kemal
Keyword(s):  
Whole Genome Sequencing ◽  
Multiplex Pcr ◽  
Genome Sequencing ◽  
Screening Method ◽  
Positive Control ◽  
Whole Genome ◽  
Rt Pcr ◽  
Pcr Screening ◽  
Single Target ◽  
Alpha Variant

Background: The ongoing COVID-19 pandemic has led to the emergence of several variants of concern. To rapidly identify those variants, screening samples for whole-genome sequencing (WGS) prioritization could be performed.  Objective: We optimized the polymerase chain reaction (PCR) screening method to identify the mutation in spike and ORF1a regions.  Methods: We adopted primers targeting mutation in spike and ORF1a region from another study. We optimized the PCR screening method using kits readily available in Indonesia. Firstly, we compared N1 and N2 primers as internal positive control. We also compared GoTaq® 1-Step RT-qPCR System and Indonesia TFRIC-19 BioCOV-19 for the multiplex reaction. We used the optimized composition to screen SARS-CoV-2 positive samples from April – June 2021. Samples with spike and/or ORF1a target failure were subjected to whole genome sequencing (WGS).  Results: The results demonstrated the N2 BioCOV-19 reaction as the optimized multiplex PCR composition for spike and ORF1a mutations screening. Whole-genome sequencing has shown that a sample with spike and ORF1a targets failure to be Alpha variant, while other samples with single target failure as non-variants of concern. Therefore, a multiplex RT-PCR composition has been optimized to detect mutation in spike and ORF1a regions. Conclusion: We have optimized a multiplex RT-PCR composition to detect mutation in spike and ORF1a regions.

scholarly journals Phenotypic H-Antigen Typing by Mass Spectrometry Combined with Genetic Typing of H Antigens, O Antigens, and Toxins by Whole-Genome Sequencing Enhances Identification of Escherichia coli Isolates

2016 ◽  
Vol 54 (8) ◽  
pp. 2162-2168 ◽  
Author(s):  
Keding Cheng ◽  
Huixia Chui ◽  
Larissa Domish ◽  
Angela Sloan ◽  
Drexler Hernandez ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Shiga Toxin ◽  
Gene Pair ◽  
Whole Genome ◽  
Genetic Typing ◽  
Content Type ◽  
O Antigens

Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinicalEscherichia coliisolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprisingwzxandwzyand that comprisingwzmandwzt. The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenicE. colityping.

scholarly journals Mass Spectrometry–Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping

2016 ◽  
Vol 62 (6) ◽  
pp. 839-847 ◽  
Author(s):  
Keding Cheng ◽  
Yi-Min She ◽  
Huixia Chui ◽  
Larissa Domish ◽  
Angela Sloan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Close Correlation ◽  
Clinical Isolates ◽  
Whole Genome ◽  
Sequence Coverage ◽  
E Coli ◽  
Reference Strains

Abstract BACKGROUND Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 μg or 1.465 × 107 cells) level and close correlation (r2 > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.

scholarly journals Molecular Investigation of an Ontario Mumps Outbreak using Whole Genome Sequencing

2017 ◽  
Vol 4 (suppl_1) ◽  
pp. S359-S359
Author(s):  
Patrick Stapleton ◽  
Alireza Eshaghi ◽  
Eddie Chong-King ◽  
Mark Cardona ◽  
Steve Masney ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Fragment Size ◽  
Pcr Amplification ◽  
Mumps Virus ◽  
Likelihood Method ◽  
Cdna Libraries ◽  
Whole Genome ◽  
Rt Pcr ◽  
Distinct Genotype

Abstract Background In early 2017 an outbreak of Mumps virus affected over 100 individuals in the province of Ontario, concurrent with multiple mumps virus outbreaks across North America. Traditional genotyping of mumps outbreaks relies on sequencing a portion of the small hydrophobic (SH) gene, but has limited capability to distinguish between strains of the same genotype. Most mumps cases in Ontario in recent years are of genotype G. We used a novel whole genome sequencing (WGS) protocol to perform a molecular epidemiological investigation of the outbreak. Methods Throat (n = 5) and buccal (n = 15) swabs positive by RT-PCR for SH or Fusion (F) gene targets were cultured in primary Rhesus monkey kidney cells. Cell free viral extract underwent RT-PCR and subsequent PCR amplification using overlapping primer pairs to cover the entire 15 kilobase (kb) genome. The first 8 samples were amplified with 18 pairs of overlapping primers, which was reduced to 9 sets (average fragment size 1.9 kb, range 1.6–2.8 kb) for the final 12 samples. Mumps cDNA libraries were prepared with Nextera XT kit and WGS of the indexed fragments was performed with V2 reagent kits on the Illumina MiSeq instrument. Reference based genome assembly was performed using samtools version 1.4. Phylogenetic analysis was performed by maximum likelihood method in MEGA7. Results We identified two distinct genotype G lineages comprised of 9 patients each and closely related to a 2009–2010 outbreak in Ontario and New York (Figure 1). Inter-lineage single nucleotide polymorphism (SNP) differences ranged from 25 to 31, whereas intra-lineage SNPs ranged from 0 to 8 SNPs. Two outlying sequences, of genotype C and G respectively, may represent sporadic introduction of virus from other areas. Time from virus isolation to SNP based analysis was approximately 4 days. Conclusion WGS of Mumps virus culture isolates using the PCR fragment method identified two distinct genotype G lineages in a large provincial outbreak. This method may aid public health authorities identify separate transmission chains in the case of large outbreaks. Disclosures All authors: No reported disclosures.

scholarly journals Rapid High Throughput Whole Genome Sequencing of SARS-CoV-2 by using One-step RT-PCR Amplification with Integrated Microfluidic System and Next-Gen Sequencing

2021 ◽  
Author(s):  
Tao Li ◽  
Hye Kyung Chung ◽  
Papa K. Pireku ◽  
Brett F. Beitzel ◽  
Mark A. Sanborn ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Pcr Amplification ◽  
Amplicon Sequencing ◽  
Microfluidic System ◽  
Full Genome Sequence ◽  
Whole Genome ◽  
Rt Pcr ◽  
Viral Isolate ◽  
One Step

The long-lasting global COVID-19 pandemic demands timely genomic investigation of SARS-CoV-2 viruses. Here we report a simple and efficient workflow for whole genome sequencing utilizing one-step RT-PCR amplification on a microfluidic platform, followed by MiSeq amplicon sequencing. The method uses Fluidigm Integrated Fluidic Circuit (IFC) and instruments to amplify 48 samples with 39 pairs of primers, including 35 custom designed primer pairs and four additional primer pairs from the ARTIC network protocol v3. Application of this method on RNA samples from both viral isolate and clinical specimens demonstrate robustness and efficiency of this method in obtaining the full genome sequence of SARS-CoV-2.

scholarly journals Investigation of Respiratory Syncytial Virus Outbreak on an Adult Stem Cell Transplant Unit by Use of Whole-Genome Sequencing

2017 ◽  
Vol 55 (10) ◽  
pp. 2956-2963 ◽  
Author(s):  
Yijun Zhu ◽  
Teresa R. Zembower ◽  
Kristen E. Metzger ◽  
Zhengdeng Lei ◽  
Stefan J. Green ◽  
...  
Keyword(s):  
Stem Cell ◽  
Respiratory Syncytial Virus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Stem Cell Transplant ◽  
Pcr Amplification ◽  
Adult Stem Cell ◽  
Cell Transplant ◽  
Whole Genome ◽  
Syncytial Virus

ABSTRACTA viral whole-genome sequencing (WGS) strategy, based on PCR amplification followed by next-generation sequencing, was used to investigate a nosocomial respiratory syncytial virus-B (RSV-B) outbreak in a hematology-oncology and stem cell transplant unit. RSV-B genomes from 16 patients and health care workers (HCWs) suspected to be involved in the outbreak were compared to RSV-B genomes that were acquired from outpatients during the same time period but epidemiologically unrelated to the outbreak. Phylogenetic analysis of the whole genome identified a cluster of 11 patients and HCWs who had an identical RSV-B strain which was clearly distinct from strains recovered from individuals unrelated to the outbreak. Sequence variation of the glycoprotein (G) gene alone was insufficient to distinguish the outbreak strains from the outbreak-unrelated strains, thereby demonstrating that WGS is valuable for local outbreak investigation.

scholarly journals Screening of triploid with low-coverage whole-genome sequencing by a single-nucleotide polymorphism-based test in miscarriage tissue

2019 ◽  
Vol 36 (12) ◽  
pp. 2525-2531
Author(s):  
Qian Geng ◽  
Xiaoli Cui ◽  
Yaqi Zhang ◽  
Lijuan Zhang ◽  
Cai Zhang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Single Nucleotide Polymorphism ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Uniparental Disomy ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Quadratic Curve ◽  
Low Coverage

Abstract Purpose To establish a single-nucleotide polymorphism-based analysis (SBA) method to identify triploidy in the miscarriage tissue by using low-coverage whole-genome sequencing (LC-WGS). Methods The method was established by fitting a quadratic curve model by counting the distribution of three heterozygous mutation content intervals. The triploid test result was mainly determined by the opening direction and the axis of symmetry of the quadratic curve, and Z test between the same batch samples was also used for auxiliary judgment. Results Two hundred thirteen diploid samples and 8 triploid samples were used for establishment of the analytical method and 203 unknown samples were used for blind testing. In the blind testing, we found 2 cases positive for triploidy. After chromosome microarray analysis (CMA) and mass spectrometry verification, we found that both samples were true positives. We randomly selected 5 samples from the negative samples for mass spectrometry verification, and the results showed that these samples were all true negatives. Conclusions Our method achieved accurate detection of triploidy in the miscarriage tissue and has the potential to detect more chromosomal abnormality types such as uniparental disomy (UPD) using a single LC-WGS approach.

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