Identification of Campylobacter jejuni, Campylobacter coli and Campylobacter lari by the nucleic acid amplification system NASB^R

Abstract

NASBAR, an isothermal amplification technique for nucleic acids, was evaluated for the specific identification of Campylobacter jejuni, Camp. coli and Camp. lari. A set of primers and a probe were chosen from the 16S rRNA sequence of Campylobacter. The probe was hybridized in solution with the amplified nucleic acids of 12 Campylobacter species and nine other Gram-negative bacteria. The probe was shown to hybridize specifically to the amplified single-stranded RNA of Camp. jejuni, Camp. coli and Camp. lari in an enzyme-linked gel assay (ELGA). In a Camp. jejuni model system the combination of NASBAR and ELGA was able to detect ca 1000 rRNA molecules. The presence of an excess of Gram-negative bacteria did not influence the sensitivity of detection. A number of 6 cfu of Camp. jejuni, present in a total count of 4 x 10(6) cfu of Gram-negative bacteria, resulted in a positive hybridization signal.

Construction Strategy for an Internal Amplification Control for Real-Time Diagnostic Assays Using Nucleic Acid Sequence-Based Amplification: Development and Clinical Application

“…The major product of a NASBA reaction is single-stranded RNA, and there are various means whereby this product can be detected to produce a NASBA signal, such as ethidium bromide staining (22), enzyme-linked gel assay (22), and electrochemiluminescence detection (25). Alternatively, NASBA products can specifically be detected in real time by using molecular beacons (20) included in the reaction mixture (13).…”

Presença de Campylobacter spp. em cortes refrigerados de frango

“…O par de iniciadores, específico para o gênero Campyobacter, empregado foi OT1559 (5'-CTGCTTAACACAAGTTGAGTAGG-3') (UYTTENDAELE et al, 1994;LÜBECK et al, 2003) e 18-1 (5'-TTCCTTAGGTACCGTCAGAA-3') (VANCAMP et al, 1993;DOCHERTY et al, 1996;LÜBECK, et al 2003). Esses iniciadores amplificam uma sequência de 287 pb do gene rRNA 16S.…”

Toward an International Standard for PCR-BasedDetection of Food-Borne Thermotolerant Campylobacters: AssayDevelopment and AnalyticalValidation

“…(20). Thus, one probe (25) and three primer sets (Table 2) targeting 16S and one primer pair targeting 23S ribosomal DNA (rDNA) (4,5,24,25) were tested on 105 Campylobacter isolates (Table 1). For testing the published PCR assays, the reaction conditions used, including temperature profile and DNA polymerase, were essentially as described in the original publications.…”