Rapid, Simple and Sensitive Detection of Q Fever by Loop-Mediated Isothermal Amplification of the htpAB Gene

Abstract

BackgroundQ fever is the most widespread zoonosis, and domestic animals are the most common sources of transmission. It is not only difficult to distinguish from other febrile diseases because of the lack of specific clinical manifestations in humans, but it is also difficult to identify the disease in C. burnetii-carrying animals because of the lack of identifiable features. Conventional serodiagnosis requires sera from the acute and convalescent stages of infection, which are unavailable at early diagnosis. Nested PCR and real-time PCR require equipment. In this study, we developed a Loop-Mediated Isothermal Amplification (LAMP) assay to identify C. burnetii rapidly and sensitively.MethodsA universal LAMP primer set was designed to detect the repeated sequence IS1111a of the htpAB gene of C. burnetii using PrimerExplorer V4 software. The sensitivity of the LAMP assay was evaluated using known quantities of recombined reference plasmids containing the targeted genes. The specificity of the developed LAMP assay was determined using 26 members of order Rickettsiae and 18 other common pathogens. The utility of the LAMP assay was further compared with real time PCR by the examination 24 blood samples including 6 confirmed and 18 probable Q fever cases, which diagnosed by IFA serological assessment and real time PCR. In addition, 126 animal samples from 4 provinces including 97 goats, 7 cattle, 18 horses, 3 marmots and 1 deer were compared by these two methods.ResultsThe limits of detection of the LAMP assay for the htpAB gene were 1 copy per reaction. The specificity of the LAMP assay was 100%, and no cross-reaction was observed among the bacteria used in the study. The positive rate of unknown febrile patients was 33.3%(95%CI 30.2%–36.4%) for the LAMP assay and 8.3%(95%CI 7.4%–9.2%) for the real time PCR(P<0.05). Similarly, the total positive rate of animals was 7.9%(95%CI 7.1%–8.7%) for the LAMP assay and 0.8%(95%CI 0.7%–0.9%)for the real time PCR(P<0.01). Using the developed LAMP assay, Q fever in the Yi Li area, Xinjiang Province, was confirmed.ConclusionsThe LAMP assay is a potential tool to support the diagnosis of Q fever in humans and domestic animals in the field, especially in the rural areas of China, because of its rapid and sensitive detection without the aid of sophisticated equipment or a complicated protocol.

Development of loop-mediated isothermal amplification assays for rapid and easy detection of Coxiella Burnetii

“…It has been applied for the detection of several rickettsial pathogens (Paris et al, 2008;Huber et al, 2012;Pan et al, 2013). The method requires a specially designed primer set that recognizes at least six independent regions of the target gene, which increases the specificity as well as the rapidity of the reaction.…”

Single Nucleotide Polymorphism Genotyping and Distribution of Coxiella burnetii Strains from Field Samples in Belgium

“…Q fever laboratory diagnosis is most commonly realized by serology and molecular biology, which rely on several techniques, such as nested PCR (8), real-time PCR (9), and more recently loop-mediated isothermal amplification (LAMP) assay (10). Different target genes have been used in order to detect C. burnetii DNA, such as the broad-spectrum 16S rRNA gene (11), the outer membrane protein-coding gene com1, and the isocitrate dehydrogenase gene icd (12) or the repetitive insertion sequence IS1111 of the htpAB gene (8,10).…”

“…Different target genes have been used in order to detect C. burnetii DNA, such as the broad-spectrum 16S rRNA gene (11), the outer membrane protein-coding gene com1, and the isocitrate dehydrogenase gene icd (12) or the repetitive insertion sequence IS1111 of the htpAB gene (8,10).…”

First report on optimization of loop-mediated isothermal amplification (LAMP) for the diagnosis of Babesia felis

“…As the small subunit (SSU) 18S rRNA gene is one of the most frequently used genes in phylogenetic studies and an important marker for random target polymerase chain reaction (PCR) in environmental biodiversity screening therefore we selected it (Makimura et al 1994). The pairs of lamp primers given in Table 1 were designed by LAMP primer designing software PrimerExplorer V4 (Pan et al 2013). Following parameters were kept in mind while designing the primers: 1.…”