scholarly journals Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

2016 ◽  
Vol 7 ◽  
Author(s):  
Dinka Mandakovic ◽  
Benjamín Glasner ◽  
Jonathan Maldonado ◽  
Pamela Aravena ◽  
Mauricio González ◽  
...  
Keyword(s):  
16S Rdna ◽  
Restriction Enzyme ◽  
Specific Detection ◽  
16S Rdna Pcr ◽  
Pcr Rflp ◽  
Piscirickettsia Salmonis ◽  
Selection For

A rapid method for identification of typical Leuconostoc species by 16S rDNA PCR-RFLP analysis

2003 ◽  
Vol 55 (1) ◽  
pp. 295-302 ◽  
Author(s):  
Jichan Jang ◽  
Bongjoon Kim ◽  
Jongho Lee ◽  
Hongui Han
Keyword(s):  
16S Rdna ◽  
Rapid Method ◽  
Rflp Analysis ◽  
16S Rdna Pcr ◽  
Pcr Rflp

scholarly journals PCR-Restriction Fragment Length Polymorphism Analysis for Identification of Bacteroides spp. and Characterization of Nitroimidazole Resistance Genes

2000 ◽  
Vol 38 (9) ◽  
pp. 3209-3213 ◽  
Author(s):  
Simon L. J. Stubbs ◽  
Jon S. Brazier ◽  
Paul R. Talbot ◽  
Brian I. Duerden
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rdna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Fragment Length Polymorphism ◽  
Rdna Sequencing ◽  
16S Rdna Pcr ◽  
Pcr Rflp ◽  
Restriction Fragment Length

Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroidesinfections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novelnim gene exhibited 75% DNA sequence similarity withnimB. These rapid, accurate, and inexpensive methods should enable improved identification ofBacteroides spp. and the detection of MTZ resistance determinants.

scholarly journals Rapid differentiation of Prevotella intermedia and P. nigrescens by 16S rDNA PCR-RFLP

1996 ◽  
Vol 44 (1) ◽  
pp. 41-43 ◽  
Author(s):  
S. E. Milsom ◽  
S. V. Sprague ◽  
D. Dymock ◽  
A. J. Weightman ◽  
W. G. Wade
Keyword(s):  
16S Rdna ◽  
Prevotella Intermedia ◽  
16S Rdna Pcr ◽  
Pcr Rflp

DETECTING MYCOBACTERIUM TUBERCULOSIS RAPIDLY AND MYCOBACTERIUM TUBERCULOSIS STRAINS WITHOUT IS6110 SEQUENCE BY PCR ASSAY

2012 ◽  
pp. 15-19
Author(s):  
Thi Chau Anh Nguyen ◽  
Hoang Bach Nguyen ◽  
Hai Duong Huynh ◽  
Nu Xuan Thanh Le ◽  
Xuan Cuong Le ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rdna ◽  
False Negative ◽  
Clinical Samples ◽  
Tuberculosis Complex ◽  
Pcr Assay ◽  
Complex Objects ◽  
Method Performance ◽  
Gene Coding ◽  
16S Rdna Pcr

Background: The Nested IS6110 PCR is used for detecting tuberculosis, however IS6110 sequence is not present in the genome of all strains of M.tuberculosis, the result may be false negative. The gene coding 16S ribosome always contains a short sequence specific to M. tuberculosis complex. Objects: Performance of the 16S Real-time PCR to detect M. tuberculosis and combining to the nested IS6110 PCR to determine the rate of Mtb strains without IS6110 from clinical samples. Materials and method: Performance of 16S rDNA PCR by commercial kit of Viet A Inc. for all 480 samples, the samples which were positive with the 16S rDNA PCR were retested in IS6110 PCR assay by in-house kit. Results: The Realtime 16S rDNA PCR detected 258 cases (53.8%) of tuberculosis. There were 3 (1.2 %) M. tuberculosis strains which do not harbor IS6110 sequence in genome. Conclusion: The IS6110 nested PCR can be applied more widely than the 16S rDNA realtime PCR. In case of using IS6110 PCR assay, results may show a low proportion of false negative. Combining 16S rDNA PCR with the IS6110 based PCR allowed detection of deletion of IS6110 sequence in M. tuberculosis isolates.

Isolation of Enterobacter sakazakii from Stable Flies, Stomoxys calcitrans L. (Diptera: Muscidae)†

2006 ◽  
Vol 69 (3) ◽  
pp. 671-673 ◽  
Author(s):  
F. MRAMBA ◽  
A. BROCE ◽  
L. ZUREK
Keyword(s):  
16S Rdna ◽  
Foodborne Pathogen ◽  
Animal Manure ◽  
Stomoxys Calcitrans ◽  
Fecal Coliforms ◽  
Stable Fly ◽  
Enterobacter Sakazakii ◽  
Stable Flies ◽  
16S Rdna Pcr ◽  
Glucose Agar

Enterobacter sakazakii is an opportunistic foodborne pathogen that causes meningitis, enterocolitis, and sepsis, primarily in immunocompromised infants. Previously, it was suggested that stable flies, Stomoxys calcitrans, were a vector or reservoir of this pathogen. In our study, by means of a culturing approach combined with 16S rDNA PCR–restriction fragment length polymorphism genotyping and sequencing, we screened 928 individual stable flies collected in Kansas and Florida. Two stable flies (0.2%) were positive for E. sakazakii. In addition, 411 (44%) stable flies carried bacteria-forming red colonies (presumably enterics) on a violet red bile glucose agar (mean count = 6.4 × 104 CFU per fly), and 120 (13%) stable flies carried fecal coliforms (mean count = 8.7 × 103 CFU per fly). Sequencing of 16S rDNA showed that enterics from violet red bile glucose agar were represented by several genera, including Escherichia, Shigella, Providencia, Enterobacter, Pantoea, Proteus, Serratia, and Morganella. Our study shows that stable flies carry bacteria typically present in animal manure (a developmental site of stable fly larvae), which indicates that the natural reservoir of E. sakazakii is the digestive tract or manure of domestic animals. The low prevalence of E. sakazakii associated with stable flies suggests that stable flies do not play a major role as a reservoir or vector of this pathogen.

scholarly journals Identification of restriction enzyme in the FSHR gene of indonesian local cattle

2021 ◽  
Vol 888 (1) ◽  
pp. 012024
Author(s):  
P W Prihandini ◽  
A Primasari ◽  
M Luthfi ◽  
D Pamungkas ◽  
A P Z N L Sari ◽  
...  
Keyword(s):  
Restriction Enzyme ◽  
Restriction Site ◽  
Follicle Stimulating Hormone ◽  
Restriction Enzymes ◽  
Future Studies ◽  
Fshr Gene ◽  
Pcr Rflp ◽  
Mapping Analysis ◽  
Pcr Products ◽  
Enzyme Mapping

Abstract The restriction enzyme is important for genotyping using the PCR-RFLP technique. Therefore, this study aims to identify the restriction enzyme mapping in the partial sequence of the follicle-stimulating hormone receptor (FSHR) gene in Indonesian local cattle. A total of 29 samples sized 306 bp, were aligned with Genbank sequence acc no. NC_032660, resulting three polymorphic sites, namely g.193G>C, g.227T>C, and g.275A>C. Furthermore, the restriction mapping analysis using the NEBcutter program V2.0 showed that no enzyme recognized the SNP g.275A>C, while the SNP g.193G>C and g.227T>C were identified by the AluI and MscI enzymes, respectively. The AluI enzyme cuts at two positions (193 bp and 243 bp) in the G allele sample producing three fragments namely 50 bp, 63 bp, and 193 bp, meanwhile, in the C allele, the AluI cuts only in position 243 bp, hence, the fragment products are 63 bp and 243 bp. In contrast, the MscI enzyme was only recognized in the T allele, producing fragments sized 77 bp and 229 bp but failed to identify the restriction site along with the PCR products in the C allele. Based on the results, the SNPs (g.193G>C and g.227T>C) and restriction enzymes (AluI and MscI) are applicable for genotyping local Indonesian cattle using the PCR-RFLP technique in future studies.

scholarly journals Melanocortin-4 Receptor (MC4R) gene polymorphism and its effect on growth traits in Madura cattle

2019 ◽  
Vol 44 (1) ◽  
pp. 38
Author(s):  
P. W. Prihandini ◽  
S. Sumadi ◽  
G. Suparta ◽  
D. Maharani
Keyword(s):  
Growth Traits ◽  
Balance Control ◽  
Direct Sequencing ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Mc4r Gene ◽  
Melanocortin 4 Receptor ◽  
Pcr Rflp ◽  
Reverse Primer ◽  
Selection For

Melanocortin-4 receptor (MC4R) gene has an important role in the regulation of feed intake and energy balance control. The objective of this study was to identify the single nucleotide polymorphisms (SNPs) of MC4R gene and their association with growth traits in Madura cattle. A total of 198 calves were used in this study.Forward primer: 5’-GTCGGGCGTCTTGTTCATC-3’and reverse primer: 5’-GCTTGTGTTTAGCATCGCGT-3’ were used to amplify approximately 493 bp of MC4R gene. The results showed that two SNPs, g.1133C>G and g.1108C>T were identified by direct sequencing. The PCR-RFLP method was performed to genotype all individuals studied based on SNP g.1133C>G, and its SNP was significantly associated with shoulder height (SH) at yearling age (P<0.05). Animals with GG genotype had a higher SH (110.35±6.40cm) than those with CC (102.00±8.00 cm) and CG genotype (105.96±6.23 cm). The SNP g.1133 C>G changed amino acid from valine to leucine. In conclusion, the SNP g.1133C>G of the MC4R gene may be used as a marker-assisted selection for SH trait in Madura cattle.

scholarly journals Minimally Invasive Detection ofPiscirickettsia salmonis in Cultivated Salmonids via the PCR

1998 ◽  
Vol 64 (8) ◽  
pp. 3066-3069 ◽  
Author(s):  
Sergio Marshall ◽  
Sekou Heath ◽  
Vitalia Henríquez ◽  
Cristián Orrego
Keyword(s):  
Minimally Invasive ◽  
Specific Detection ◽  
Microbial Pathogen ◽  
Less Invasive ◽  
Piscirickettsia Salmonis ◽  
Fish Serum ◽  
The Way

ABSTRACT The attributes of the PCR allowed implementation of an assay for specific detection of Piscirickettsia salmonis from a few microliters of fish serum. This opens the way to less invasive modes of sampling for this microbial pathogen in salmonids.

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