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

Podsumowanie

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

Streszczenie

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.

Wprowadzenie

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 left untreated^1,2. Therefore, early diagnosis to guide an appropriate treatment is critical for patient care. Polymerase chain reaction (PCR) and quantitative real time PCR (qPCR) assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples^3-5. Both PCR and qPCR are costly and often not readily available in resource-constrained areas for routine work.

Originally described by Notomi⁶, loop-mediated isothermal amplification (LAMP) offers an alternative DNA amplification method. LAMP uses Bst DNA polymerase for strand displacement DNA synthesis along with specially designed primer sets that recognize at least six independent regions of the target gene. The most significant advantage of LAMP is that amplification occurs under isothermal conditions. Therefore, only a water bath, heating block or an incubator is required. This method has been used to detect several rickettsial pathogens^7-9. Visualization of amplified DNA products by gel electrophoresis is the most accurate method which can differentiate true positives from false positives due to nonspecific amplification. However the procedures involved in gel electrophoresis are not practical for resource-limited areas. Several alternative methods were developed to detect the reaction products. These alternative, such as turbidity derived from magnesium pyrophosphate formation¹⁰ or using a fluorescent intercalating dye to be visualized under UV light^11,12 are more favorable than running a gel. The LAMP reagents were stable for one month when stored at 25 °C and 37 °C, which are ambient temperatures in tropical and sub-tropical countries where Q fever is endemic¹³.

A LAMP assay was developed in our laboratory to detect the presence of C. burnetii in plasma samples¹⁴. Here we describe a simple protocol for the preparation of the LAMP reaction mixture from the lyophilized reagents. The lyophilized reagents are stable for one month when stored at RT. When combined with an easy visualization method, this is an ideal method to use for detecting C. burnetii in a resource-limited setting.

Protokół

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, equating to 5 x 10⁹ copies (34.2 x 10^-9 g / 4.1 x 10⁶ g x 6.02 x 10²³) of DNA.
3. Add 10 µl of plasmid DNA (5 x 10⁹ copies/µl) to 90 µl of water for a 10-fold dilution to make a DNA sample of 5 x 10⁸ copies/µl.
4. Repeat step 1.3 to prepare DNA samples of 5 x 10⁷, 5 x 10⁶, 5 x 10⁵, 5 x 10⁴, 5 x 10³, 5 x 10², 5 x 10¹, and 5 x 10⁰ copies/µl .

2. Prepare DNA Template from Samples for LAMP Reaction

1. Perform DNA extraction from human plasma samples using a commercial DNA mini kit according to manufacturer's protocol. Use a total of 200 µl sample for extraction and elute extracted DNA in a 20 µl volume.

3. Prepare 2x LAMP Reaction Buffer

1. Mix 200 µl of 10x Pol Buffer, 320 µl of 5 M trimethylglycine, 12 µl of 1 M MgSO₄, 280 µl of dNTP mixture (10 mM each) and 188 µl of water to make 1 ml of 2x LAMP buffer. Mix by pulse-vortexing for 10 sec.

4. Perform Standard LAMP Reaction

1. Mix 12.5 µl of 2x LAMP buffer (as prepared in step 3; 40 mM Tris-HCl (pH 8.8), 20 mM KCl, 16 mM MgSO₄, 20 mM (NH₄)₂SO₄, 0.2% Triton X-100, 1.6 M trimethylglycine, 1.4 mM dNTP mixture), 1.2 µl of primer mix, 1 µl of Bst DNA polymerase (8 U/µl), and 5.3 µl of water to make reaction mixture (20 µl) in a 0.2 ml PCR tube.
2. Add 5 µl of DNA (from step 1 or 2) to the 20 µl reaction mixture and mix well.
3. Close tube and incubate at 60 °C for 60 min in a water bath or heating block. This is the optimal reaction temperature determined previously.
4. Add 5 µl of 10x gel loading buffer to terminate the reaction.
5. Load 5 µl of reaction products to a 2% agarose gel stained with intercalating nucleic acid stain.
6. Run gel at 100 V for 35 min in 1x TBE buffer.
7. Visualize the results by UV light.

5. Perform LAMP Reaction with Reconstituted Reagents

1. Mix 125 µl of 10x Pol Buffer, 200 µl of 5 M trimethylglycine, 7.5 µl of 1 M MgSO₄, 667.5 µl of water to make 1 ml of reconstitution buffer. Mix by pulse-vortexing for 10 sec.
2. Remove tubes which contain the lyophilized reagents from the sealed aluminum foil bag. Do not use the tubes if the aluminum foil bag is not sealed or if it is damaged.
3. Add 20 µl of reconstitution buffer into each tube to re-suspend lyophilized reagents.
4. Mix by pipetting buffer up and down 5 times. Take a look to make sure the lyophilized reagents are completely re-suspended. The white powder should disappear in the tube.
5. Add 5 µl of DNA template (from step 1 or 2) to the reconstituted reagents and mix well.
6. Close tube and incubate at 60 °C for 60 min in a water bath or heating block.
7. Add 5 µl of 10x gel loading buffer to terminate the reaction.
8. Load 5 µl of reaction products to a 2% agarose gel stained with intercalating nucleic acid stain.
9. Run gel at 100 V for 35 min in 1x TBE buffer.
10. Visualize the results by UV light.

6. Perform LAMP Reaction with Reconstitution Buffer Containing HNB or Fluorescent Intercalating Dye

1. Mix 125 µl of 10x Pol Buffer, 200 µl of 5 M trimethylglycine, 7.5 µl of 1 M MgSO₄, 7.5 µl of 20 mM HNB (or 12.5 µl of 100x fluorescent intercalating dye) and 660 µl (or 655 µl) of water to make 1 ml of reconstitution buffer. Mix by pulse-vortexing for 10 sec.
2. Use the reconstituted buffer prepared in section 6 to repeat the steps 5.2 - 5.6.
3. Visualize the results by the naked eye (reaction containing HNB) or a UV light (reaction containing fluorescent intercalating dye).

7. Perform Real-time LAMP Reaction with Tube Scanner

1. Add 5 µl DNA to the reconstituted reagents containing fluorescent intercalating dye, close tube and insert it to the tube scanner.
2. Set incubation temperature at 60 °C. Measure the fluorescence at 520 nm for 60 min.

Wyniki

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 th...

Dyskusje

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...

Materiały

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