A highly sensitive quantitative real-time PCR assay based on the groEL gene of contemporary Thai strains of Orientia tsutsugamushi

D.H Paris,N Aukkanit,K Jenjaroen,S.D Blacksell,N.P.J Day

doi:10.1111/j.1469-0691.2008.02671.x

Abstract

Partial nucleotide sequences (459 bp) of the groEL gene (encoding the 60-kDa heat shock protein, HSP60) from 23 contemporary isolates of Orientiatsutsugamushi isolated from patients with acute scrub typhus in Thailand were compared with 16 reference strain sequences to evaluate the potential of groEL as a conserved and representative target for molecular diagnostics‥ Overall nucleotide identity within all available O. tsutsugamushi isolates (n = 39) was 98.8% (range: 95.0–100), reflecting a high degree of conservation; nucleotide identities were 67.5% and 65.6%, respectively, when typhus and spotted fever group rickettsiae were included‥ A highly sensitive and quantitative real-time PCR assay was designed and evaluated using 61 samples, including buffy coats from patients in Thailand and Laos. Reliable and accurate quantitation of bacterial loads allows further investigation of other diagnostic methods and may lead to an improved understanding of the pathophysiology of acute scrub typhus, an important but under-recognized disease.

Highlights

The Rickettsiaceae family consists of a group of highly fastidious, obligate intracellular Gram-negative organisms
Comparison of percentage identities of groEL gene sequences among antigenic groups showed that Thai strains and non-Thai strains shared a mean nucleotide identity of 96.7%
When Thai O. tsutsugamushi strains were compared with typhus group (TG) and spotted fever group (SFG) strains, the percentage identity levels were found to be 67.5% and 65.6%, with similar values for non-Thai Orientia strains when compared to TG and SFG strains, i.e. 67.2% and 65.7%, respectively (Table 3)

Summary

Introduction

The Rickettsiaceae family consists of a group of highly fastidious, obligate intracellular Gram-negative organisms. They are divided into three groups, based on antigenic reactivity—the scrub typhus group, typhus group (TG) and spotted fever group (SFG). The development of rapid, inexpensive and accurate diagnostic methods is necessary, both to improve diagnosis and to promote awareness of these potentially serious but treatable diseases in highly populous rural areas of Southeast Asia. With the increasing availability of gene sequences, allowing the exploitation of more gene-based targets, molecular assays have been developed and evaluated for the diagnosis of acute scrub typhus. A common target gene used in nested conventional as well as real-time PCR assays, encodes the 56-kDa outer membrane protein [5,6]. Another target gene encodes the 47-kDa outer membrane protein, used in a real-time PCR assay [7]

Methods

Results

Conclusion