Rapid, simple, and sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification

Lei Pan,Qinghui Liu,Guiqiang Wang,Lijuan Zhang

doi:10.1186/1471-2334-12-254

Abstract

BackgroundSpotted fever caused spotted fever group rickettsiae (SFGR) is prevalent throughout China. In this study, we describe a rapid, simple, and sensitive loop-mediated isothermal amplification (LAMP) assay targeting the ompB gene of spotted fever group rickettsiae ideal for application in China. The LAMP assay has the potential to detect spotted fever group rickettsiae early in infection and could therefore serve as an alternative to existing methods.MethodsA set of universal primers which are specific 7 common species of spotted fever group rickettsiae in China were designed using PrimerExplorer V4 software based on conserved sequences of ompB gene. The sensitivity, specificity and reproducibility of the LAMP were evaluated. The LAMP assay for detecting SFGR was compared with conventional PCR assays for sensitivity and specificity in early phase blood samples obtained from 11 infected human subjects.ResultsThe sensitivity of the LAMP assay was five copies per reaction (25 μL total volume), and the assay did not detect false-positive amplification across 42 strains of 27 members of the order Rickettsiales and 17 common clinical pathogens. The LAMP assay was negative to typhus group rickettsiae including R. prowazekii and R. typhi for no available conserved sequences of ompB was obtained for designing primers.To evaluate the clinical applicability of the LAMP assay, a total of 11 clinical samples, 10 samples confirmed serologically (3 cases), ecologically (1 case), by real-time polymerase chain reaction (PCR; 2 cases), ecologically and by real-time PCR (1 case), and serologically and by real-time PCR (3 cases) were analyzed by the ompB LAMP assay. Data were validated using a previously established nested PCR protocol and real-time PCR. A positive LAMP result was obtained for 8 of the 10 confirmed cases (sensitivity, 73%; specificity, 100%), while none of these samples were positive by nested PCR (sensitivity, 0%; specificity, 100%).ConclusionsThe LAMP assay described here is the most reliable among the three methods tested and would be an ideal choice for development as a rapid and cost-effective means of detecting SFGR in China.

Highlights

Spotted fever caused spotted fever group rickettsiae (SFGR) is prevalent throughout China
Specificity of loop-mediated isothermal amplification (LAMP) To determine the specificity of the LAMP assay for the detection of SFGR, 27 members of the order Rickettsiales one of the SFGR assignments was made by analysis of bacterial cultures individually, the remaining three were confirmed individually by indirect fluorescent antibody analysis (IFA) of acute- and convalescent-phase serum samples, two were confirmed individually by real-time polymerase chain reaction (PCR), one was confirmed by both analysis of bacterial cultures and real-time PCR, and three were confirmed by both IFA of acute- and convalescent- phase serum samples and real-time PCR
To evaluate the utility of LAMP for use with clinical samples, the 11 samples analyzed by IFA were subjected to analysis by LAMP and nested PCR (Table 4)

Summary

Introduction

Spotted fever caused spotted fever group rickettsiae (SFGR) is prevalent throughout China. The LAMP assay has the potential to detect spotted fever group rickettsiae early in infection and could serve as an alternative to existing methods. Spotted fever is caused by obligate (Gram-negative) intracellular spotted fever group rickettsiae (SFGR) [1]. Unrecognized pathogens, combined with the lack of a rapid and sensitive diagnostic method for identifying SFGR, may be responsible for causing delayed treatment or misdiagnosis that could lead to the development of severe disease and fatality [7]. SFGR can be isolated and reliably identified from samples cultivated in the laboratory, this approach for early diagnoses has its drawbacks, as this method is generally associated with low isolation rates and requires sophisticated and expensive equipment. Other methods that have been applied for this purpose, such as those involving the polymerase chain reaction (PCR) (i.e., conventional, nested, and realtime PCR), require expensive, specialized instruments those are not widely available

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