Development of Single Nucleotide Polymorphism (SNP)-Based Triplex PCR Marker for Serotype-specific Escherichia coli Detection

Rahman, Md-Mafizur; Lim, Sungtaek; Park, Yung Chul

doi:10.3390/pathogens11020115

Cited by 9 publications

(11 citation statements)

References 63 publications

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“…To date, several molecular approaches have been applied to detect Salmonellae , such as PFGE [ 39 ], phage typing [ 40 ], and multilocus sequence typing (MLST) [ 41 ], but they have some limitations. However, SNP-based molecular techniques have recently been proposed as a cost-effective identification method of various bacterial species, including, Salmonella [ 42 ], E. coli [ 43 ]; Mycobacterium [ 44 ]. The SNPs have discrimination power for comparing of bacterial subspecies and at the serovar level using different bioinformatics software [ 45 ].…”

Section: Discussionmentioning

confidence: 99%

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Genome-Wide Searching Single Nucleotide-Polymorphisms (SNPs) and SNPs-Targeting a Multiplex Primer for Identification of Common Salmonella Serotypes

2022

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A rapid and high-quality single-nucleotide polymorphisms (SNPs)-based method was developed to improve detection and reduce salmonellosis burden. In this study, whole-genome sequence (WGS) was used to investigate SNPs, the most common genetic marker for identifying bacteria. SNP-sites encompassing 15 sets of primers (666–863 bp) were selected and used to amplify the target Salmonella serovar strains, and the amplified products were sequenced. The prevalent Salmonella enterica subspecies enterica serovars, including Typhimurium; Enteritidis, Agona, enterica, Typhi, and Abony, were amplified and sequenced. The amplified sequences of six Salmonella serovars with 15 sets of SNP-sites encompassing primers were aligned, explored SNPs, and SNPs-carrying primers (23 sets) were designed to develop a multiplex PCR marker (m-PCR). Each primer exists in at least two SNPs bases at the 3′ end of each primer, such as one was wild, and another was a mismatched base by transition or transversion mutation. Thus, twenty-three sets of SNP primers (242–670 bp), including 13 genes (SBG, dedA, yacG, mrcB, mesJ, metN, rihA/B, modA, hutG, yehX, ybiY, moeB, and sopA), were developed for PCR confirmation of target Salmonella serovar strains. Finally, the SNPs in four genes, including fliA gene (S. Enteritidis), modA (S. Agona and S. enterica), sopA (S. Abony), and mrcB (S. Typhimurium and S. Typhi), were used for detection markers of six target Salmonella serotypes. We developed an m-PCR primer set in which Salmonella serovars were detected in a single reaction. Nevertheless, m-PCR was validated with 21 Salmonella isolates (at least one isolate was taken from one positive animal fecal, and n = 6 reference Salmonella strains) and non-Salmonella bacteria isolates. The SNP-based m-PCR method would identify prevalent Salmonella serotypes, minimize the infection, and control outbreaks.

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Section: Discussionmentioning

confidence: 99%

“…LT2 (NC-003197) ( Table S1 ). According to our published article, SNP-based primers and protocols were developed [ 43 ]. We used the MUMer package (v3.19, NUCmer algorithm, National Institutes of Health, Bethesda, MD, USA) to investigate the SNP sites on the WGS [ 61 ].…”

Section: Methodsmentioning

confidence: 99%

Genome-Wide Searching Single Nucleotide-Polymorphisms (SNPs) and SNPs-Targeting a Multiplex Primer for Identification of Common Salmonella Serotypes

2022

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“…The second issue is their inability to detect single‐nucleotide polymorphisms (SNPs) or other alterations/mutations due to their low resolution and precision. However, it is possible to examine localized sequence alterations (usually 3–6 nucleotides) using allele‐specific primers or fluorophore‐labeled probes, however, the exact base change and the precise coordinate cannot be obtained with high confidence 97 . The limitations of these techniques can be well tackled using advanced biosensors which include electrochemical, and microfluidic‐based platforms, which provide cost‐effective, ultra‐highly sensitive, and selective detection of target analyte within a limited time frame.…”

Section: Recent Advances In Microbial Sensingmentioning

confidence: 99%

Internet‐of‐medical‐things integrated point‐of‐care biosensing devices for infectious diseases: Toward better preparedness for futuristic pandemics

Parihar

Yadav

Sadique

et al. 2023

Bioengineering & Transla Med

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Microbial pathogens have threatened the world due to their pathogenicity and ability to spread in communities. The conventional laboratory‐based diagnostics of microbes such as bacteria and viruses need bulky expensive experimental instruments and skilled personnel which limits their usage in resource‐limited settings. The biosensors‐based point‐of‐care (POC) diagnostics have shown huge potential to detect microbial pathogens in a faster, cost‐effective, and user‐friendly manner. The use of various transducers such as electrochemical and optical along with microfluidic integrated biosensors further enhances the sensitivity and selectivity of detection. Additionally, microfluidic‐based biosensors offer the advantages of multiplexed detection of analyte and the ability to deal with nanoliters volume of fluid in an integrated portable platform. In the present review, we discussed the design and fabrication of POCT devices for the detection of microbial pathogens which include bacteria, viruses, fungi, and parasites. The electrochemical techniques and current advances in this field in terms of integrated electrochemical platforms that include mainly microfluidic‐ based approaches and smartphone and Internet‐of‐things (IoT) and Internet‐of‐Medical‐Things (IoMT) integrated systems have been highlighted. Further, the availability of commercial biosensors for the detection of microbial pathogens will be briefed. In the end, the challenges while fabrication of POC biosensors and expected future advances in the field of biosensing have been discussed. The integrated biosensor‐based platforms with the IoT/IoMT usually collect the data to track the community spread of infectious diseases which would be beneficial in terms of better preparedness for current and futuristic pandemics and is expected to prevent social and economic losses.

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“…Several research groups have recently developed single nucleotide polymorphisms (SNP)-based genotyping approaches, e.g. , to investigate Mycobacterium species ( Kim et al, 2021 ; Napier et al, 2020 ), attribute hosts for Chlamydia psittaci ( Vorimore et al, 2021 ) and Campylobacter coli ( Jehanne et al, 2020 ), distinguish Rickettsia typhi from different continents ( Kato et al, 2022 ), identify Escherichia coli of specific serotype ( Rahman, Lim & Park, 2022 ), and track the spread of drug resistance in Plasmodium falciparum infections ( Jacob et al, 2021 ).…”

Section: Introductionmentioning

confidence: 99%

minSNPs: an R package for the derivation of resolution-optimised SNP sets from microbial genomic data

Hoon

Holt

Auburn

et al. 2023

PeerJ

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Here, we present the R package, minSNPs. This is a re-development of a previously described Java application named Minimum SNPs. MinSNPs assembles resolution-optimised sets of single nucleotide polymorphisms (SNPs) from sequence alignments such as genome-wide orthologous SNP matrices. MinSNPs can derive sets of SNPs optimised for discriminating any user-defined combination of sequences from all others. Alternatively, SNP sets may be optimised to determine all sequences from all other sequences, i.e., to maximise diversity. MinSNPs encompasses functions that facilitate rapid and flexible SNP mining, and clear and comprehensive presentation of the results. The minSNPs’ running time scales in a linear fashion with input data volume and the numbers of SNPs and SNPs sets specified in the output. MinSNPs was tested using a previously reported orthologous SNP matrix of Staphylococcus aureus and an orthologous SNP matrix of 3,279 genomes with 164,335 SNPs assembled from four S. aureus short read genomic data sets. MinSNPs was shown to be effective for deriving discriminatory SNP sets for potential surveillance targets and in identifying SNP sets optimised to discriminate isolates from different clonal complexes. MinSNPs was also tested with a large Plasmodium vivax orthologous SNP matrix. A set of five SNPs was derived that reliably indicated the country of origin within three south-east Asian countries. In summary, we report the capacity to assemble comprehensive SNP matrices that effectively capture microbial genomic diversity, and to rapidly and flexibly mine these entities for optimised marker sets.

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Development of Single Nucleotide Polymorphism (SNP)-Based Triplex PCR Marker for Serotype-specific Escherichia coli Detection

Cited by 9 publications

References 63 publications

Genome-Wide Searching Single Nucleotide-Polymorphisms (SNPs) and SNPs-Targeting a Multiplex Primer for Identification of Common Salmonella Serotypes

Genome-Wide Searching Single Nucleotide-Polymorphisms (SNPs) and SNPs-Targeting a Multiplex Primer for Identification of Common Salmonella Serotypes

Internet‐of‐medical‐things integrated point‐of‐care biosensing devices for infectious diseases: Toward better preparedness for futuristic pandemics

minSNPs: an R package for the derivation of resolution-optimised SNP sets from microbial genomic data

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