Name: the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii
Uploaded: 2016-04-18T14:00:00
Description: Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

This research was supported by Naval Medical Research Center, research work unit 6000.RAD1.J.A0310. The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, or the U.S. Government. Wei-Mei Ching is an employee of the U.S. Government. This work was prepared as part of her official duties. Title 17 U.S.C. &#167;105 provides that &#39;Copyright protection under this title is not available for any work of the United States Government.&#39; Title 17 U.S.C. &#167;101 defines a U.S. Government work as a work prepared by military service member or employee of the U.S. Government as part of that person&#39;s official duties.

1. Prepare Plasmid DNA Dilutions as Standard for LAMP Reaction
<ol>
	<li>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 &#181;g/&#181;l of DNA.</li>
	<li>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). T...

<ol>
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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...

The small Gram-negative bacterium Coxiella burnetii is the causative agent of Q fever, which is a worldwide zoonosis. Due to Q fever&#39;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 untreated1,2. 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 samples3-5. 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 pathogens7-9. 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 light11,12 are more favorable than running a gel. The LAMP reagents were stable for one month when stored at 25 &#176;C and 37 &#176;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.

<table border="0" cellpadding="0" cellspacing="0" width="773">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">LAMP primers</td>
			<td style="width:188px;">Eurofins MWG operon</td>
			<td style="width:131px;">10 nmol, salt free</td>
			<td style="width:240px;">Sequence designed by customer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Bst DNA polymerase</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">M0275L</td>
			<td style="width:240px;">8 units/ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Thermo pol buffer</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">B9004S</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">dNTP mixture</td>
			<td style="width:188px;">New England Biolabs</td>
			<td style="width:131px;">N0447L</td>
			<td style="width:240px;">10 mM each</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Betaine</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">B0300</td>
			<td style="width:240px;">5 M, Trimethylglycine</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Magnesium Sulfate</td>
			<td style="width:188px;">Sigma-Aldrich</td>
			<td style="width:131px;">M3409-1ML</td>
			<td style="width:240px;">1 M</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">10X Bluejuice</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">10816-015</td>
			<td style="width:240px;">gel loading buffer</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">SYBR green</td>
			<td style="width:188px;">Invitrogen</td>
			<td style="width:131px;">S7585</td>
			<td style="width:240px;">10,000X, visualize products in tubes</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">GelRed</td>
			<td style="width:188px;">Phenix Research Products</td>
			<td style="width:131px;">RGB-4103</td>
			<td style="width:240px;">10,000X, visualize products in gels</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Lyophilized reagents</td>
			<td style="width:188px;">Gene Reach</td>
			<td style="width:131px;"></td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Hydroxynaphthol blue</td>
			<td style="width:188px;">Fluka</td>
			<td style="width:131px;">33936-10G</td>
			<td style="width:240px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">ESEQuant tube scanner</td>
			<td style="width:188px;">Qiagen</td>
			<td style="width:131px;"></td>
			<td style="width:240px;">real-time detection</td>
		</tr>
	</tbody>
</table>

the development of lyophilized loop-mediated isothermal amplification reagents for the detection of coxiella burnetii

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 &#176;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.

The lyophilized LAMP reagents inside the 0.2 ml tube contains Bst DNA polymerase, primers, and dNTPs. 20 &#181;l 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...

Watch this Scientific Journal Video about The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii at JoVE.com

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

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

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

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

The overall goal of this experiment is to develop a simple LAMP method using lyophilized reagents for the detection of C.burnetii in patient samples. The main advantage of this protocol is that no coaching is required for the storage of the reagents. This method is ideal for use in resource-limited settings where Q fever is endemic.Demonstrating the procedure will be Tatiana, a technician from our laboratory. Beginning with DNA for the target gene IS1111a from C.burnetii RSA493, determine the plasmid concentration with a spectrophotometer at 260 nanometers. Convert the plasmid DNA concentration into copy number using the following calculations, where 6330 equals the length and base pairs of the plasmid, and 34.2 nanograms per microliter is the concentration of the plasmid, as just determined using the spectrophotometer.Then prepare 8-to 10-fold serial dilutions of the plasmid to arrive at the following number of copies per microliter. To prepare the DNA template for the LAMP reaction, begin with 200 microliters of human plasma samples, and with the DNA miniprep kit, extract the DNA according to the manufacturer's protocol. Dilute the final sample into a 20-microliter volume.Prepare 1 mililiter of 2x LAMP reaction buffer by combining the following reagents. Mix by pulse-vortexing for 10 seconds. To perform the standard LAMP reaction, in a 0.2 milliliter PCR tube, mix 12.5 microliters of 2x LAMP buffer, 1.2 microliters of primer mix, 1 microliter of Bst DNA polymerase, and 5.3 microliters of water.Next, add 5 microliters of DNA to the 20 microliter reaction and mix well, then incubate at 60 degrees Celsius in a water bath or heating block for 60 minutes. After the incubation, terminate the reaction by adding 5 microliters of 10x gel loading buffer to the tube. Then load 5 microliters of the reaction products onto a 2%agarose gel stained with intercalating nucleic acid stain.Run the gel in 1x TBE at 100 volts for 35 minutes, then use UV light to visualize the results. To perform the LAMP reaction with reconstituted reagents, prepare 1 milliliter of reconstitution buffer by combining the following reagents. Mix by pulse-vortexing for 10 seconds.Next, remove the tubes that contained the lyophilized reagents from the sealed aluminium foil bag. For Step 4.2, it is very important to make sure the aluminium foil bag is properly sealed before opening it. Do not use the tubes if the aluminum bag is not sealed or is damaged.Add 20 microliters of reconstitution buffer to each tube of reagents and mix by pipetting up and down five times. Ensure that the lyophilized reagents are completely re-suspended. Then, add 5 microliters of DNA template to the reconstituted reagents and mix well.Incubate at 60 degrees Celsius in a water bath or heating block for 60 minutes. To terminate the reaction after the incubation, add 5 microliters of 10x gel loading buffer, then add 5 microliters of the reaction products onto a 2%agarose gel stained with intercalating nucleic acid stain. Run the gel in 1x TBE buffer at 100 volts for 35 minutes, then visualize the results by UV light.To perform the LAMP reaction with hydroxynaphthol blue, or HNB, or intercalating dye, prepare 1 milliliter of reconstitution buffer by combining the following reagents that include HNB or a fluorescent intercalating dye. Mix by pulse-vortexing for 10 seconds. Carry out the LAMP reaction as just described and use UV light or the naked eye to visualize the results.To perform the LAMP reaction with a tube scanner, add 5 microliters of DNA to the reconstituted reagents containing the fluorescent intercalating dye, then insert the closed tube into the tube scanner. Set the incubation temperature to 60 degrees Celsius and measure the fluorescence at 520 nanometers for 60 minutes. This figure shows the LAMP reaction results on agarose gels with freshly-prepared reagents and lyophilized reagents.Observe the lyophilized reagent maintains its activity. LAMP reactions prepared by both reagents detected 25 copies of DNA template. In this figure, the detection of the reaction's products with HNB or fluorescent intercalating dye in the reaction is shown.The addition of HNB to the reaction produces a visual color change, from purple to blue, with 25, 50, and 100 copies of DNA template present in the reactions. In addition, the inclusion of fluorescent intercalating dye in the reaction shows a strong fluorescent signal with UV light when similar DNA copy numbers are present. Shown here are the results from using the tube scanner to monitor the fluorescent signals in real-time with fluorescent intercalating dye.The fluorescent signals are above baseline after 14, 18, 21, and 23 minutes, with 10^6, 10^4, 10^3, and 100 copies of DNA template in the reactions. Once mastered, this procedure can be done in 90 minutes. After its development, this assay paved the way for quick diagnosis of Coxiella burnetii infection in resource-limited areas.After watching this video, you should have a good understanding of how to reconstitute lyophilized LAMP reagents. Don't forget that patient samples can be infectious and safety precautions should always be taken while performing this procedure.

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