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Articles by R.K. Agarwal
Total Records ( 4 ) for R.K. Agarwal
  K. Porteen , R.K. Agarwal and K.N. Bhilegaonkar
  A polymerase chain reaction was standardized for the identification of Aeromonas sp. using primers against 16S rRNA gene and aerolysin gene. The primers used were found to be highly specific for Aeromonas sp. and did not give positive result with other bacteria including other Gram positive and Gram-negative bacteria. The minimum detection level of PCR was found to be 102 and 104 cells mL-1 in case of 16S rRNA and aerolysin gene targeted assay, respectively. Suitability of the enrichment broth (Alkaline peptone water-cephalothin, APW-C) when tested to detect Aeromonas from the spiked samples gave good results on direct usage of the broth for template preparation without any subsequent treatment. The kinetics of the spiking study indicated that a minimum of 24 h enrichment was required for the detection of Aeromonas by cultural and PCR method. Among two PCR assays detection limits achieved by PCR targeting16S rRNA gene were better than aerolysin gene PCR assay. The results were comparable to cultural method. A total of 100 samples comprising of 50 each of chicken and fish samples were screened by cultural and PCR methods for the presence of Aeromonas. Two chicken samples and three fish samples turned out to be positive by both cultural method and PCR targeting 16S rRNA. From this study it was concluded that PCR assay targeting 16S rRNA gene can be used for the rapid detection of Aeromonas from chicken and fish samples after one step enrichment in APW-C.
  M. Habtamu Taddele , R. Rathore , K. Dhama and R.K. Agarwal
  The present study was conducted to evaluate the discriminating power of RAPD-PCR and PCR-RFLP to characterize Salmonella gallinarum (S. gallinarum) isolates. Fifteen S. gallinarum isolates of poultry origin recovered from various sources in different regions of India including three reference strains were characterized using RAPD-PCR and PCR-RFLP. RAPD-PCR, using 5 primers (NSC I, NSC II, NSC III, URP-6 and primer 1290), grouped S. gallinarum isolates into 4-6 RAPD types. The discrimination power (D) of NSC II, URP-6 and primer 1290 was 0.848, 0.81 and 0.695, respectively and 0.62 for NSC I and NSC III. The phylogenetic dendrogram classified the isolates based on their geographic location. However, most S. gallinarum isolates were clustered closely together. In comparative analysis NSC II primer was placed in a separate cluster. The PCR amplification of the 16s rRNA and fimH genes produced approximately 572 bp and 1008 bp product, respectively, for all S. gallinarum isolates. Restriction Endonuclease (RE) enzymes viz., EcoRI, SmaI, AluI, BgiII, MspI and HaeIII and SalI, AluI, MspI and HaeIII were used to digest PCR products of 16s rRNA and fimH genes, respectively. These restriction enzymes generated 2-4 bands of varying molecular size ranging from 40 to 410 bp and 50 to 620 bp for 16s rRNA and fimH genes, respectively. AluI targeted against the fimH gene was the only enzyme which classified S. gallinarum isolates into three groups. In conclusion, the RAPD-PCR profile proved to be used as an epidemiological typing tool since it classified the S. gallinarum isolates according to their geographic origin.
  P.S. Bagalakote , R.S. Rathore , T.P. Ramees , H.V. Mohan , M. Sumankumar , R.K. Agarwal , A. Kumar and K. Dhama
  In modern years, Arcobacters are reflected as potential emerging food-borne zoonotic entero-pathogens. Arcobacter species displayed a wide variety of genetic diversity. The study was carried out to genotype and find molecular heterogeneity of Arcobacter spp. (Arcobacter butzleri, A. cryaerophilus and A. skirrowii), isolated from different sources from Bareilly region, Uttar Pradesh, India by using randomly amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR). RAPD-PCR was performed using genomic DNA of Arcobacter isolates (n = 56; 33 A. butzleri, 20 A. cryaerophilus, 3 A. skirrowii; recovered from chicken meat, pork, sheep faeces, goat faeces, poultry intestinal contents and human diarrhoeal stool samples) as template by employing two published primers. The RAPD profiling for primer 1 (HLWL85) yielded number of bands rangeing between 2-8 (500-3100 bp). Out of 56 isolates, 54 showed bands giving a typeability of 96.4%. These 54 typable strains were grouped to 35 types and giving discriminatory power of 0.9762. Primer 2 (OPA-11) yielded RAPD-PCR profiles comprising of 2-7 bands (210-2800 bp). Out of total 56 isolates, 54 were typable with a discriminatory power of 0.9336. This is the first report from India regarding RAPD profiling of Arcobacter spp. This study reveals epidemiological relationship of Arcobacter isolate from various sources and will help to design suitable prevention and control strategies for this important pathogen having public health significance.
  Manoj Jinu , R.K. Agarwal , B. Sailo , M.A. Wani , Ashok Kumar , K. Dhama and M.K. Singh
  The aim of the study was to compare Polymerase Chain Reaction (PCR) and conventional method for detection of Salmonella from field poultry samples (n = 510, poultry blood and faeces 255 each). The prevalence rate of Salmonella in chicken was found to be 5.09% using conventional method and 5.88% by PCR assay. Serotyping of 26 Salmonella isolates revealed 57.69% Salmonella Typhimurium, 19.23% rough type, 15.38% Salmonella Enteritidis and 7.69% untypable. Among Salmonella Typhimurium isolates, 73.33% were from poultry blood and 26.66% from faeces samples. All isolates belonging to Typhimurium and Enteritidis serotypes were confirmed by PCR targeting of Salmonella Typhimurium (typh) and Salmonella Enteritidis (ent) specific genes. However, 4 isolates found to be rough type also turned out to be positive for ent gene. The PCR employed for detection of Salmonella was found 100% sensitive for poultry blood but its sensitivity was very less (77.77%) for faeces samples as compared with culture method. However, PCR was 100% specific with regard to faeces samples. The specificity from blood samples was 97.89% by PCR. The positive predictive values of PCR from blood and faecal samples were 77.27 and 100% with a concordance of 98.03 and 99.21%, respectively. The negative predictive values from blood and faecal samples were 100 and 99.19%. The study demonstrated usefulness of genus specific PCR for detection of Salmonella in poultry clinical samples. Owing to its robustness and rapidity it can be used for wide epidemiological studies. Serotype specific PCR detection of Typhimurium and Enteritidis serotypes has added advantage in identifying them even where there is loss of O antigen.
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