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.

Fully Automated Lab-On-A-Disc Platform for Loop-Mediated Isothermal Amplification Using Micro-Carbon-Activated Cell Lysis

“…The results indicated that the time required for the LAMP chamber to reach a temperature of 60 °C was only 32 s (standard deviation of 2.1705). Furthermore, Figure 9 shows that the temperature in the LAMP chamber was maintained between 57 and 63 °C, which is the temperature range required for a stable reaction [ 15 , 29 ].…”

Real-time PCR biochip for on-site detection of Coxiella burnetii in ticks

“…Detection of C. burnetii using loop-mediated isothermal amplification (LAMP) assay [ 43 , 44 ] is rapid and comparable to real-time PCR. The positive detection is based on a change of colour in the reaction mix after 30 min incubation.…”

“…Therefore, the UR-qPCR proposed in this study has the advantages of being rapid, with less than 20 min reaction time, and sensitive, at 100% sensitivity compared to other real-time PCR systems. The UR-qPCR and the crude DNA preparation [ 44 ] together will take less than 30 min for the detection of C. burnetii on-site.…”

Real-time PCR Biochip for On-Site Detection of Coxiella Burnetii in Ticks

“…Therefore, the UR-qPCR proposed in this study has the advantages of being rapid, with less than 20 min reaction time, and sensitive, at 100% sensitivity compared to other realtime PCR systems. The UR-qPCR and the crude DNA preparation [39] together will take less than 30 min for the detection of C. burnetii on-site.…”

“…Therefore, UR-qPCR could be used for on-site conformational diagnosis of Q fever, for the prompt control of milk, blood, or serum samples. Detection of C. burnetii using loop-mediated isothermal ampli cation (LAMP) assay [38,39] is rapid and comparable to real-time PCR. The positive detection is based on change of colour in the reaction mix after 30 min incubation.…”