The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Summary

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

Abstract

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 °C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

Introduction

The small Gram-negative bacterium Coxiella burnetii is the causative agent of Q fever, which is a worldwide zoonosis. Due to Q fever's worldwide distribution and the high infectivity of C. burnetii, US military and civilian personnel deployed overseas are at risk of being infected in the endemic areas.

Q fever manifests in two forms: acute and chronic infections. Acute Q-fever presents itself with flu-like symptoms, hepatitis, or pneumonia, and is usually a self-limiting disease with a low mortality rate. Chronic Q-fever, while less prevalent, often results in endocarditis, which has a much higher mortality rate if lef....

Protocol

1. Prepare Plasmid DNA Dilutions as Standard for LAMP Reaction

1. Use a spectrophotometer to measure the optical density (O.D.) at 260 nm to determine the plasmid (containing target gene IS1111a of C. burnetii RSA 493) DNA concentration. An O.D.₂₆₀ of 1 equates to 50 µg/µl of DNA.
2. Convert the amount of plasmid DNA into the copy number. Since the size of the plasmid is 6,330 bp, the molecular weight of this plasmid is 4.1 x 10⁶ g (6,330 bp x 649 g/bp). The DNA concentration of the plasmid is 34.2 ng/µl (as determined from step 1.1). 1 µl of this DNA sample contains 34.2 ng of plasmid DNA, eq....

Results

The lyophilized LAMP reagents inside the 0.2 ml tube contains Bst DNA polymerase, primers, and dNTPs. 20 µl of reconstitution buffer is used to re-suspend the lyophilized reagents. Figure 1 shows the LAMP reaction results on agarose gels with freshly prepared reagents and lyophilized reagents. The lyophilized reagent does not reduce its activity. LAMP reactions prepared by both reagents can detect 25 copies of DNA template. Figure 2 shows the det.......

Discussion

Previously, we developed a sensitive and specific LAMP assay targeting the insertion element IS1111a¹⁴. The IS1111 element was selected because it is highly conserved among the different strains of C. burnetii and its high number of copies (7 to 110) in the bacteria (Klee 2006). Our results showed that LAMP can detect about 25 copies of the IS1111 element, which may correlate to as little as one chromosomal copy of Coxiella DNA. In this study, Bst DNA polym.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
LAMP primers	Eurofins MWG operon	10 nmol, salt free	Sequence designed by customer
Bst DNA polymerase	New England Biolabs	M0275L	8 units/ml
10x Thermo pol buffer	New England Biolabs	B9004S
dNTP mixture	New England Biolabs	N0447L	10 mM each
Betaine	Sigma-Aldrich	B0300	5 M, Trimethylglycine
Magnesium Sulfate	Sigma-Aldrich	M3409-1ML	1 M
10x Bluejuice	Invitrogen	10816-015	gel loading buffer
SYBR green	Invitrogen	S7585	10,000x, visualize products in tubes
GelRed	Phenix Research Products	RGB-4103	10,000x, visualize products in gels
Lyophilized reagents	Gene Reach
Hydroxynaphthol blue	Fluka	33936-10G
ESEQuant tube scanner	Qiagen		real-time detection