Development of a Rapid and Sensitive Colorimetric Loop-Mediated Isothermal Amplification Assay: A Novel Technology for the Detection of Coxiella burnetii From Minimally Processed Clinical Samples

Abstract

Q fever is an important zoonotic disease caused by the bacterium Coxiella burnetii. The agent is considered as a potential agent for bioterrorism because of its low infectious dose, aerial route of transmission, resistance to drying, and many commonly used disinfectants. Humans are largely infected by the inhalation of aerosols that are contaminated with parturition products of infected animals as well as by the consumption of unpasteurized milk products. Thus, rapid and accurate detection of C. burnetii in shedders, especially those that are asymptomatic, is important for early warning, which allows controlling its spread among animals and animal-to-human transmission. In the present study, a colorimetric loop-mediated isothermal amplification (LAMP) assay was developed to confirm the presence of IS1111a gene of C. burnetii in sheep vaginal swabs. The sensitivity of this assay was found to be very comparable to the quantitative PCR (qPCR) assay, which could detect three copies of the gene, which corresponds to a single cell of C. burnetii. The applicability of the colorimetric LAMP assay in the disease diagnosis was assessed by evaluating 145 vaginal swab samples collected from the sheep breeding farms with a history of stillbirth and repeated abortions. Compared to qPCR, colorimetric LAMP had a sensitivity of 93.75% (CI, 69.77-99.84%) and specificity of 100% (CI, 97.20-100%), with a positive (PPV) and negative predictive value (NPV) of 100 and 99.24%, respectively. A very high level of agreement was observed between both colorimetric LAMP and reference qPCR assay. The colorimetric LAMP assay reported here is a rapid and simple test without extensive sample preparation and has a short turnaround time of <45 min. To the best of our understanding, it is the very first study describing the use of colorimetric LAMP assay that detects C. burnetii in vaginal swab samples with minimal sample processing for DNA extraction.

Keywords: Coxiella burnetii; POC; Q fever; colorimetric LAMP; qPCR; sensitive detection.

Figure 1

Nucleotide sequence of the transposase gene insertion element IS1111a of C. burnetii used for designing the LAMP primers. Numbers on the left correspond to the positions in the transposase gene (GenBank, accession no. AE016828.2). The locations of the target sequences are underlined with an arrow. FIP consists of the F1c and F2 sequences, and BIP consists of B1c and B2 sequences.

Figure 2

Comparison of the sensitivity of colorimetric LAMP, conventional PCR and qPCR. (A) Results of colorimetric LAMP analysis, (B) Agarose gel electrophoresis of colorimetric LAMP products, (C) Agarose gel electrophoresis of conventional PCR products (D) Results of qPCR Analysis, (E) Standard curve and amplification efficiency, (F) Melting curve analysis. Lane, M: DNA marker; Lanes 1-9: Reaction results from a 10-fold serial dilution of plasmid containing the transposase gene from 3 × 10⁷ to 3 × 10⁻¹ copies per reaction; Lane 10: Negative control (nuclease free water).