Development of a Loop-Mediated Isothermal Amplification Assay for Rapid, Sensitive and Specific Detection of a <i>Campylobacter jejuni</i> Clone

Abstract

AbSTRAcT. Loop-mediated isothermal amplification (LAMP) assay is a simple, rapid and specific detection method and has been used for detection and identification of different Campylobacter species. In this study, we develop a LAMP assay specific for detection of a particular clone (clone SA) of Campylobacter jejuni, associated with the vast majority of recent sheep abortions in the U.S. Using a set of specific primers for C. jejuni IA3902 (a clone SA isolate) and genomic DNA or boiled cell extract as template, the target DNA was amplified at 63°C for 50 min in a water bath. A positive reaction was identified visually as white precipitate or fluorescence under UV, and confirmed by gel electrophoresis. Detection limit of the assay was comparable to that of conventional PCR. The LAMP was shown to be specific for detection of clone SA when tested on a number of C. jejuni strains of different genetic backgrounds. Applicability of the LAMP assay for specific detection of clone SA was demonstrated in animal tissues experimentally infected with IA3902 or genetically diverse C. jejuni strains. Since clone SA is the predominant cause of sheep abortions in the U.S. and is a zoonotic pathogen, the LAMP assay may be a valuable detection tool in future epidemiological studies. Rapid and reliable detection and identification of Campylobacter from clinical and environmental sources is of paramount significance in epidemiological studies. Several methods such as culture, biochemical assays and PCR-based assays have been developed for detection and identification of Campylobacter species [5,9,11,12,14]. Despite being effective and accurate, the bacterial culture and biochemical assays may require up to 4 days and thus are time-consuming. PCR-based assays are more rapid to identify Campylobacter species than bacterial culture and biochemical assays, but the requirement of sophisticated procedures and expensive equipments to detect the DNA targets may greatly restrict their application in clinical settings.Recently, a rapid and simple assay named loop-mediated isothermal amplification (LAMP) [6] for the detection of C. jejuni [16,18], Campylobacter coli [16,18] and Campylobacter fetus [17,19] has been developed. The LAMP assay is a highly specific strand displacement amplification method, using 4 to 6 different primers designed to 8 from 6 distinct regions of the target DNA sequence. Only one efficient DNA polymerase (Bst DNA polymerase) is required, the amplification reaction proceeds under a single and isothermal temperature (i.e., 60-65°C), and it has a high amplification efficiency. Moreover, the LAMP assay synthesizes a large amount of by-product, an insoluble white precipitate of magnesium pyrophosphate, which can be judged visually by the appearance of a white precipitate, or fluorescence observed under UV with the addition of SYBR Green dye. The amplification reaction does not require a complex thermocycler; rather a simple water bath is sufficient for the entire process [3,6,15].The objective of the current study was to de...

RNAseq Reveals Complex Response of Campylobacter jejuni to Ovine Bile and In vivo Gallbladder Environment

“…Verification of complete removal of any contaminating DNA was performed via PCR amplification of a portion of the CjSA_1356 gene, which is part of the capsule locus and has previously been determined via comparative genomics to only be present in C. jejuni IA3902, using primers SA1356F, and SA1356R (Luo et al, 2012). A single 24 h in vivo sample failed to isolate any RNA following extraction and purification; therefore, it did not continue with the rest of the library preparation.…”

Wide but Variable Distribution of a Hypervirulent Campylobacter jejuni Clone in Beef and Dairy Cattle in the United States

“…In this study, initial preliminary identification of clone SA was done with a rapid PCR method that targets CJSA_1356, which is one of the variable genes in the capsule locus and is quite specific for clone SA isolates. Previous work has shown the utility of this PCR method for initial screening for clone SA isolates ( 37 ). However, this method is not 100% specific for clone SA, which requires further confirmation of the putative clone SA isolates by other methods.…”

“…The second primer pair (mapA-F, 5′-GAG TGC TTG TGC AAC TAA AC-3′, and mapA-R, 5′-ATA GCA TCT TGA GTT GCT CC-3′) was specific for C. jejuni ( 51 ). The third PCR primer pair (CJSA_1356F, 5′-TCC CAT TTG GAT GTT GTT GA-3′, and CJSA_1356R, 5′-CAG AAC CTG GCC ACA AAC TT-3′) was used for identification of putative C. jejuni clone SA as described previously ( 37 ). C. jejuni IA3902, a clinical isolate of clone SA, was used as a positive control for the PCR, whereas reactions with no DNA template were used as negative controls.…”

High Prevalence of Fluoroquinolone-Resistant Campylobacter Bacteria in Sheep and Increased Campylobacter Counts in the Bile and Gallbladders of Sheep Medicated with Tetracycline in Feed

“…DNA template was extracted from Campylobacter colonies using single-cell lysis buffer (57). Primers specific for C. jejuni clone SA were designed as described previously (58). C. jejuni IA3902, a clinical isolate of clone SA, was used as the positive control, while a reaction without template DNA served as a negative control.…”