scholarly journals DETECTION OF CAMPYLOBACTER SPP. WITH REAL-TIME POLYMERASE CHAIN REACTION

2019 ◽  
pp. 3-7
Author(s):  
G. S. Skitovich ◽  
K. V. Serova ◽  
N. B. Shadrova ◽  
O. V. Pruntova
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Carbon Dioxide Concentration ◽  
Ion Concentration ◽  
Animal Origin ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Polymerase Chain

Bacteria of Campylobacter genus are ones of the main zoonotic pathogens causing human and animal diseases. Campylobacter organisms are microaerophiles and, therefore, require low oxygen concentration (3–5%) and high carbon dioxide concentration (3–10%) for their growth. They use amino acids rather than carbons as a source of energy. Classical bacteriological methods for Campylobacter spp. detection are not always successful due to diffi culties in creating optimal conditions for their growth. Therewith, development and implementation of molecular methods for Campylobacter detection and identifi cation are of current importance. Assay for qualitative Campylobacter spp. detection with real-time polymerase chain reaction using CFX-96 thermocycler was optimized. Highly specifi c segment of 16S rRNA gene allowing identifi cation of 6 Campylobacter species: C. jejuni, C. coli, C. lari, C. upsaliensis, C. helveticus и C. hyointestinalis was selected as an amplifi cation target. Optimal magnesium ion concentration (2.5 мМ) and primer annealing temperature (58 °С) were determined. Eighteen reference strains of various bacteria were tested. Only tests of Campylobacter genus strains gave positive results. The method sensitivity was 40 target molecules. The said method was used for testing 76 samples of raw materials of animal origin. Campylobacter spp. genome was detected in 18 samples. Obtained results showed that the optimized variant of real-time polymerase chain reaction based on 16S rRNA gene amplifi cation was a specifi c, sensitive, rapid, reproducible and accurate method for qualitative detection of Campylobacter spp. in samples of raw animal materials.

scholarly journals Evaluasi Deteksi Berbasis PCR untuk Bakteri Bifidobacterium longum dalam Sampel Feses Bayi dari Kota Manado

2020 ◽  
Vol 20 (1) ◽  
pp. 18
Author(s):  
Beivy Jonathan Kolondam
Keyword(s):  
Polymerase Chain Reaction ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Detection Method ◽  
Bifidobacterium Longum ◽  
Local Alignment ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Search Tool ◽  
Polymerase Chain

Bifidobakteria merupakan mikroflora yang umum hidup dalam usus manusia sejak bayi. Peran Bifidobacterium longum yang positif sebagai salah satu bakteri yang menunjang kesehatan inangnya membuat bakteri ini menjadi objek studi yang menarik. Salah satu instrumen dalam penelitian adalah adalah metode deteksi bakteri B. longum yang berbasis PCR (Polymerase Chain Reaction) gen 16S rRNA. Dengan mempertimbangkan bahwa perancangan primer untuk deteksi ini sudah lebih dari 20 tahun, penelitian ini bertujuan mengevaluasi hasil deteksi melalui PCR terhadap B. longum dalam feses bayi. Akurasi hasil dilihat melalui sekuensing terhadap hasil PCR sampel yang terdeteksi positif. Dua sampel feses bayi di Manado yang diperiksa menunjukkan hasil positif dan produk PCR tersebut dilakukan sekuensing. Panjang DNA yang nyata dari hasil deteksi ini yaitu 829 bp dan bukan 831 bp. Sekuens DNA kedua sampel ini identik satu sama lain. Hasil BLAST (Basic Local Alignment Search Tool) mengonfirmasi kesamaan 100% (identik) dari kedua specimen dari Kota Manado dengan sekuens gen 16S rRNA specimen bakteri B. longum yang telah ada dalam GenBank.Kata-kata kunci: Bifidobacterium longum, Polymerase Chain Reaction, deteksi, feses, bayi. Evaluation of PCR-Based Detection for Bifidobacterium longum in Infant Fecal Samples from Manado City ABSTRACTBifidobacteria are common members of the gut microflora of humans since infant. The Bifidobacterium longum has positive roles and one of supportive bacteria to the host, which made interesting as a study object. One instrument in studying this bacterial species is the detection method based on PCR of 16S rRNA gene. In consideration of the design of primers for this detection method is already more than 20 years, this research aimed to evaluate the PCR-based detection of B. longum in infant feces. The accuracy of the method was evaluated from sequencing of DNA fragment from positive results. Two fecal samples in Manado City shown positive result were sent for sequencing. The actual length of DNA amplified by PCR was 829 bp, not 831 bp. The DNA sequence of both samples were identical to each other. The BLAST (Basic Local Alignment Search Tool) result confirmed the similarity of both samples from Manado with 16S rRNA gene sequence of B. longum specimens in GenBank.Keywords: Bifidobacterium longum, Polymerase Chain Reaction, detection, feces, infant.

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