Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi

Quintaes, Bianca Ramalho; Leal, Nilma Cintra; Reis, Eliane Moura Falavina dos; Hofer, Ernesto

doi:10.1590/s0037-86822004000200006

Cited by 13 publications

(12 citation statements)

References 22 publications

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“…These investigators demonstrated that RAPD-PCR using primer 1254 did not discriminate among Salmonella isolates. However, this is in contrast with the report of Quintanes et al (2004) which recorded highest discriminatory power amongst clinical Salmonella isolates using Primers 784 and 1254 in Brazil.…”

Section: Discussioncontrasting

confidence: 99%

Serotyping and molecular typing of Salmonella species isolated from wastewater in Nsukka, Nigeria

Dickson¹,

Chibuogwu²,

Ezeonu³

2016

Afr. J. Microbiol. Res.

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The objective of this investigation was to isolate and identify Salmonella serovars present in wastewater from the University of Nigeria, Nsukka (UNN) wastewater treatment plant and to evaluate the sensitivity and precision of different microbial typing methods (conventional and molecular), in identifying and characterizing Salmonella species. A total of 100 suspected Salmonella colonies on selective media (Salmonella-Shigella agar and MacConkey Agar) were subjected to biochemical testing. A total of 12 biochemically typical Salmonella isolates were identified and further characterized. Serotyping analysis further identified 3 (25%) of the isolates as Salmonella enterica serovar Limete. Salmonella specific (16S) polymerase chain reaction (PCR) assay validated the result obtained by serotyping, although 2 of the isolates could not be serotyped and were identified as rough strains. PCR assay produced positive amplifications of 574 bp of the 16S rRNA gene specific for Salmonella, while non-Salmonella serovars were negative (100%). Random amplified polymorphic DNA (RAPD-PCR) analysis revealed the genetic relatedness of Salmonella serovars isolated from wastewater. Primers 787 and RAPD2 identified 4 RAPD-binding patterns, while primer 1254 did not give any discriminatory pattern. Molecular analyses (16S PCR and RAPD) showed discriminatory power, reproducibility, easy interpretation and performance. It is therefore a promising alternative method for typing Salmonella species.

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Section: Discussioncontrasting

confidence: 99%

Serotyping and molecular typing of Salmonella species isolated from wastewater in Nsukka, Nigeria

Dickson¹,

Chibuogwu²,

Ezeonu³

2016

Afr. J. Microbiol. Res.

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“…Oligonucleotides of short sequence were used 21,22 and the PCR reactions were prepared as stated above. Primer details and PCR conditions are shown in Table 2.…”

Section: Rapd-pcrmentioning

confidence: 99%

Incidence of non-typhoidal Salmonella in poultry products in the North West Province, South Africa

Olobatoke¹,

Mulugeta²

2015

S. Afr. J. Sci.

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This study was conducted to evaluate the incidence of non-typhoidal Salmonella (NTS) serotypes in raw and ready-to-eat (RTE) broiler products in the North West Province of South Africa. A total of 120 raw broiler samples, 40 samples of polonies and 20 samples of smoked viennas were obtained from retail points in major cities and towns in the province. Samples were subjected to aerobic plate count and later screened for the presence of NTS using phenotypic and genotypic techniques. The average bacterial count in raw products was 3.1 x 10 5 cfu/g whereas bacterial contamination of RTE products was 1.8 x 10 3 cfu/g. The average recovery rate of NTS species from raw broiler products was 12.5% and the serotypes identified were S. typhimurium (46.4%), S. enteritidis (30.9%) and S. newport (22.9%). No NTS was recovered from the RTE products. However, S. typhimurium was the predominant serotype in whole carcasses whereas S. enteritidis and S. newport were prevalent in chicken parts. Out of the 160 presumptive NTS isolates screened by polymerase chain reaction (PCR), 140 (87.5%) were confirmed for the presence of the Salmonella-specific invA gene. In addition, 115 (82.4%) of the confirmed isolates harboured the plasmid spvC gene. Random Amplified Polymorphic DNA (RAPD) fingerprinting of isolates using RAPD 1 and RAPD 3 primers, revealed some inter-and intra-serotype genetic diversity among isolates, suggesting varying sources of contamination. The results of this study represent the first report on the incidence and prevalent serotypes of NTS in chicken products in the North West Province of South Africa.

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“…Annually, there are more than 17 million cases of typhoid fever, with around 600,000 deaths (41). Genetic diversity among serovar Typhi strains has been studied extensively using molecular techniques such as pulsed-field gel electrophoresis (14,16,20,35,36), ribotyping (7,17,22), IS200 typing (37), amplified fragment length polymorphism analysis (21), and random amplification of polymorphic DNA analysis (26,33). A major drawback of these techniques is that the relationships derived from the data they provide do not necessarily reflect the true evolutionary relationships of the isolates.…”

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confidence: 99%

Single-Nucleotide-Polymorphism Typing and Genetic Relationships of Salmonella enterica Serovar Typhi Isolates

Octavia

Lan

2007

J Clin Microbiol

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Salmonella enterica serovar Typhi is a clone with a low level of variation. We developed a molecular typing method for serovar Typhi using 38 genome-wide single-nucleotide polymorphisms (SNPs) as markers detected by PCR-restriction enzyme digestion. The 73 worldwide serovar Typhi isolates studied were separated into 23 SNP profiles and four distinct genetic groups. Serovar Typhi isolates expressing the unique flagellar antigen z66 were found to cluster together and branch off from the ancestral group, suggesting that serovar Typhi was initially monophasic with only an H1 antigen and subsequently gained the z66 antigen. Typing using the 38 SNPs gave a discriminatory power of 0.87, and a minimum of 16 SNPs may be used to achieve the same level of differentiation. The SNP typing method we developed will be a valuable tool for global epidemiology studies of serovar Typhi.Typhoid fever, a serious systemic disease, is caused by Salmonella enterica serovar Typhi and is endemic in countries where problems of hygiene and sanitation remain unresolved. Annually, there are more than 17 million cases of typhoid fever, with around 600,000 deaths (41). Genetic diversity among serovar Typhi strains has been studied extensively using molecular techniques such as pulsed-field gel electrophoresis (14,16,20,35,36), ribotyping (7,17,22), IS200 typing (37), amplified fragment length polymorphism analysis (21), and random amplification of polymorphic DNA analysis (26, 33). A major drawback of these techniques is that the relationships derived from the data they provide do not necessarily reflect the true evolutionary relationships of the isolates.Two population genetics studies showed that serovar Typhi is a highly homogenous clone. Multilocus enzyme electrophoresis of 24 metabolic enzymes revealed only two major electrophoretic types (27), and multilocus sequence typing (MLST) of seven housekeeping genes found only three base substitutions in a total of 3,336 bp analyzed and divided 26 serovar Typhi isolates into four sequence types (13). Therefore, there is insufficient variation for either multilocus enzyme electrophoresis or MLST to be useful for the determination of relationships among isolates or for global epidemiological studies.To facilitate global epidemiology studies and to establish the evolutionary relationships within the serovar Typhi clone, there is a need for a molecular method that is cheap, discriminative, simple, and reproducible for the large-scale typing of isolates. Single-nucleotide polymorphisms (SNPs) are potential markers and have been used to type several pathogens, including Escherichia coli O157:H7 (42), Bacillus anthracis (25), Mycobacterium tuberculosis (8, 11), and Yersinia pestis (1). The discovery of SNPs is facilitated by the sequencing of more than one genome from the same clone. The completed genome sequences of serovar Typhi strains CT18 and Ty2 (4, 24) allowed us to explore the differences between them and to identify SNPs suitable for typing. We selected 37 SNPs that could be differentiated by ...

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Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi

Cited by 13 publications

References 22 publications

Serotyping and molecular typing of Salmonella species isolated from wastewater in Nsukka, Nigeria

Serotyping and molecular typing of Salmonella species isolated from wastewater in Nsukka, Nigeria

Incidence of non-typhoidal Salmonella in poultry products in the North West Province, South Africa

Single-Nucleotide-Polymorphism Typing and Genetic Relationships of Salmonella enterica Serovar Typhi Isolates

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