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). The DNA concentration of the plasmid is 34.2 ng/&#181;l (as determined from step 1.1). 1 &#181;l 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.</li>
	<li>Add 10 &#181;l of plasmid DNA (5 x 109 copies/&#181;l) to 90 &#181;l of water for a 10-fold dilution to make a DNA sample of 5 x 108 copies/&#181;l.</li>
	<li>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/&#181;l .</li>
</ol>
2. Prepare DNA Template from Samples for LAMP Reaction
<ol>
	<li>Perform DNA extraction from human plasma samples using a commercial DNA mini kit according to manufacturer&#39;s protocol. Use a total of 200 &#181;l&#160;sample for extraction and elute extracted DNA in a 20 &#181;l volume.</li>
</ol>
3. Prepare 2x&#160;LAMP Reaction Buffer
<ol>
	<li>Mix 200 &#181;l of 10x&#160;Pol Buffer, 320 &#181;l of 5 M trimethylglycine, 12 &#181;l of 1 M MgSO4, 280 &#181;l of dNTP mixture (10 mM each) and 188 &#181;l of water to make 1 ml of 2x LAMP buffer. Mix by pulse-vortexing for 10 sec.</li>
</ol>
4. Perform Standard LAMP Reaction
<ol>
	<li>Mix 12.5 &#181;l 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 &#181;l of primer mix, 1 &#181;l of Bst DNA polymerase (8 U/&#181;l), and 5.3 &#181;l of water to make reaction mixture (20 &#181;l) in a 0.2 ml PCR tube.</li>
	<li>Add 5 &#181;l of DNA (from step 1 or 2) to the 20 &#181;l reaction mixture and mix well.</li>
	<li>Close tube and incubate at 60 &#176;C for 60 min in a water bath or heating block. This is the optimal reaction temperature determined previously.</li>
	<li>Add 5 &#181;l of 10x gel loading buffer to terminate the reaction.</li>
	<li>Load 5 &#181;l of reaction products to a 2% agarose gel stained with intercalating nucleic acid stain.</li>
	<li>Run gel at 100 V for 35 min in 1x TBE buffer.</li>
	<li>Visualize the results by UV light.</li>
</ol>
5. Perform LAMP Reaction with Reconstituted Reagents
<ol>
	<li>Mix 125 &#181;l of 10x Pol Buffer, 200 &#181;l of 5 M trimethylglycine, 7.5 &#181;l of 1 M MgSO4, 667.5 &#181;l of water to make 1 ml of reconstitution buffer. Mix by pulse-vortexing for 10 sec.</li>
	<li>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.</li>
	<li>Add 20 &#181;l of reconstitution buffer into each tube to re-suspend lyophilized reagents.</li>
	<li>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.</li>
	<li>Add 5 &#181;l of DNA template (from step 1 or 2) to the reconstituted reagents and mix well.</li>
	<li>Close tube and incubate at 60 &#176;C for 60 min in a water bath or heating block.</li>
	<li>Add 5 &#181;l of 10x gel loading buffer to terminate the reaction.</li>
	<li>Load 5 &#181;l of reaction products to a 2% agarose gel stained with intercalating nucleic acid stain.</li>
	<li>Run gel at 100 V for 35 min in 1x TBE buffer.</li>
	<li>Visualize the results by UV light.</li>
</ol>
6. Perform LAMP Reaction with Reconstitution Buffer Containing HNB or Fluorescent Intercalating Dye
<ol>
	<li>Mix 125 &#181;l of 10x Pol Buffer, 200 &#181;l of 5 M trimethylglycine, 7.5 &#181;l of 1 M MgSO4, 7.5 &#181;l of 20 mM HNB (or 12.5 &#181;l of 100x fluorescent intercalating dye) and 660 &#181;l (or 655 &#181;l) of water to make 1 ml of reconstitution buffer. Mix by pulse-vortexing for 10 sec.</li>
	<li>Use the reconstituted buffer prepared in section 6 to repeat the steps 5.2 - 5.6.</li>
	<li>Visualize the results by the naked eye (reaction containing HNB) or a UV light (reaction containing fluorescent intercalating dye).</li>
</ol>
7. Perform Real-time LAMP Reaction with Tube Scanner
<ol>
	<li>Add 5 &#181;l DNA to the reconstituted reagents containing fluorescent intercalating dye, close tube and insert it to the tube scanner.</li>
	<li>Set incubation temperature at 60 &#176;C. Measure the fluorescence at 520 nm for 60 min.</li>
</ol>

<ol>
	<li>Anderson, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+management+of+Q+fever+United+States,+2013:+recommendations+from+CDC+and+the+Q+Fever+Working+Group.">Diagnosis and management of Q fever United States, 2013: recommendations from CDC and the Q Fever Working Group.</a> MMWR Recomm. Rep. 62 (3), 1-30 (2013).</li><li>Rolain, J. M., Boulos, A., Mallet, M. N., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+ratio+of+serum+doxycycline+concentration+to+MIC+and+rapid+decline+of+antibody+levels+during+treatment+of+Q+fever+endocarditis.">Correlation between ratio of serum doxycycline concentration to MIC and rapid decline of antibody levels during treatment of Q fever endocarditis.</a> Antimicrob. Agents and Chemother. 49 (7), 2673-2676 (2005).</li><li>Fournier, P. E., Raoult, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+PCR+and+serology+assays+for+early+diagnosis+of+acute+Q+fever.">Comparison of PCR and serology assays for early diagnosis of acute Q fever.</a> J. Clin. Microbiol. 41 (11), 5094-5098 (2003).</li><li>Schneeberger, P. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+PCR+with+serum+samples+is+indispensable+for+early+diagnosis+of+acute+Q+fever.">Real-time PCR with serum samples is indispensable for early diagnosis of acute Q fever.</a> Clin. Vaccine Immunol. 17 (2), 286-290 (2010).</li><li>Turra, M., Chang, G., Whybrow, D., Higgins, G., Qiao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+acute+Q+fever+by+PCR+on+sera+during+a+recent+outbreak+in+rural+South+Australia.">Diagnosis of acute Q fever by PCR on sera during a recent outbreak in rural South Australia.</a> Ann. N Y Acad. Sci. 1078, 566-569 (2006).</li><li>Notomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Res. 28 (12), (2000).</li><li>Paris, D. H., Blacksell, S. D., Newton, P. N., Day, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+rapid+and+sensitive+detection+of+Orientia+tsutsugamushi.+by+loo-isothermal+DNA+amplification.">Simple, rapid and sensitive detection of Orientia tsutsugamushi. by loo-isothermal DNA amplification.</a> Trans. R. Soc. Trop. Med. Hyg. 102 (12), 1239-1246 (2008).</li><li>Huber, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+assay+targeting+the+47-kDa+gene+of+Orientia+tsutsugamushi.:+A+rapid+and+sensitive+alternative+to+real-time+PCR.">Loop-mediated isothermal amplification assay targeting the 47-kDa gene of Orientia tsutsugamushi.: A rapid and sensitive alternative to real-time PCR.</a> J. Med. Microb. Diagn. 1 (112), (2012).</li><li>Pan, L., Zhang, L., Wang, G., Liu, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+sensitive+detection+of+the+ompB+gene+of+spotted+fever+group+rickettsiae+by+loop-mediated+isothermal+amplification.">Rapid, sensitive detection of the ompB gene of spotted fever group rickettsiae by loop-mediated isothermal amplification.</a> BMC Infect. Dis. 12 (254), (2012).</li><li>Mori, Y., Nagamine, K., Tomita, N., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+loop-mediated+isothermal+amplification+reaction+by+turbidity+derived+from+magnesium+pyrophosphate+formation.">Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.</a> Biochem. Biophys. Res. Commun. 289 (1), 150-154 (2001).</li><li>Qiao, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+rapid+detection+of+Bacillus+anthracis.spores.">Loop-mediated isothermal amplification for rapid detection of Bacillus anthracis.spores.</a> Biotechnol. Lett. 29 (12), 1939-1946 (2007).</li><li>Tomita, N., Mori, Y., Kanda, H., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nat. Protoc. 3 (5), 877-882 (2008).</li><li>Thekisoe, O. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+Loop-Mediated+Isothermal+Amplification+(LAMP)+reagents+and+its+amplification+efficiency+on+crude+trypanosome+DNA+templates.">Stability of Loop-Mediated Isothermal Amplification (LAMP) reagents and its amplification efficiency on crude trypanosome DNA templates.</a> J. Vet. Med. Sci. 71 (4), 471-475 (2009).</li><li>Chen, H. W., Ching, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+loop-mediated+isothermal+amplification+assays+for+rapid+and+easy+detection+of+Coxiella+Burnetii.">Development of loop-mediated isothermal amplification assays for rapid and easy detection of Coxiella Burnetii.</a> J. Microbiol Methods. 107, 176-181 (2014).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comparison+of+In-House+Real-Time+LAMP+Assays+with+a+Commercial+Assay+for+the+Detection+of+Pathogenic+Bacteria.">A Comparison of In-House Real-Time LAMP Assays with a Commercial Assay for the Detection of Pathogenic Bacteria.</a> Molecules. 20 (6), 9487-9495 (2015).</li></ol>

The authors have nothing to disclose.

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 polymerase, LAMP primers, and dNTPs were lyophilized and packaged in a zipped aluminum foil bag. The sensitivity of these lyophilized reagents remains at 25 copies; similar to the freshly prepared reagents for at least one month when stored at RT. It is very important to make sure the aluminum foil bag is properly sealed before opening it. Do not use the tubes if the aluminum foil bag is not sealed or if it is damaged. This may cause the rehydration of the lyophilized reagents which leads to the loss of its reactivity. The other critical step is the re-suspension of the lyophilized reagents with reconstitution buffer. The LAMP reaction will not work properly with reagents that are not fully re-suspended. Because of the high salt concentrations of the reaction buffer and high viscosity of the trimethylglycine solution, they cannot be successfully lyophilized with Bst DNA polymerase, LAMP primers, and dNTPs. A special step is required to prepare the reconstitution buffer with those components. Potentially the LAMP reaction can be tested at low salt concentrations with low or no trimethylglycine. If there is a reaction condition that has the same detection sensitivity but with low salt and low trimethylglycine concentration, the lyophilized reagent only needs to be reconstituted with water. This will further simplify the reaction steps.
Recently, Wang et al.15 compared twelve in-house LAMP assays with the commercialized isothermal Master Mix kit and found the in-house assays have false-positive results. In our laboratory, we have developed several LAMP assays for the detection of different bacteria and have never found false-positive results in these in-house assays. Furthermore, in the beginning stages of the development of the LAMP method, the assays were tested with both in-house prepared master mix and commercialized master mix and no difference was observed in the assays&#39; sensitivity and specificity.
Our study also presents several methods that not only can detect the LAMP products without opening the reaction tubes to decrease the likelihood of laboratory cross contamination but also have a shorter process time to read results compare to gel electrophoresis method. The advantage of using these detection methods will greatly enhance our ability to quickly perform the assay and diagnose an individual with C. burnetii infection in resource-limited areas. This will allow rapid and appropriate treatment of acute Q fever and lessen the potential of future chronic infection.
Unlike PCR-based methods where a thermocycler is needed, a heating block, water bath or incubator maintaining a constant temperature around 60 &#176;C is all that is required for the LAMP assay. This makes the assay particularly suitable in resource-limited setting. Unlike the original LAMP assay, Bst DNA polymerase, LAMP primers, and dNTPs need to be stored at -20 &#176;C. These lyophilized reagents can be stored at room temperature which makes it even more useful for a resource-limited setting where Q fever is endemic. The long term stability study of the lyophilized reagents at 4 &#176;C, 25 &#176;C, and 37 &#176;C is currently ongoing. In the future, we would like to demonstrate that this LAMP assay using lyophilized reagents can detect C. burnetii with sensitivity similar to that of PCR in a remote setting.

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><tbody><tr><td>LAMP primers</td><td>Eurofins MWG operon</td><td>10 nmol, salt free</td><td>Sequence designed by customer</td></tr><tr><td>Bst DNA polymerase</td><td>New England Biolabs</td><td>M0275L</td><td>8 units/ml</td></tr><tr><td>10x Thermo pol buffer</td><td>New England Biolabs</td><td>B9004S</td><td></td></tr><tr><td>dNTP mixture</td><td>New England Biolabs</td><td>N0447L</td><td>10 mM each</td></tr><tr><td>Betaine</td><td>Sigma-Aldrich</td><td>B0300</td><td>5 M, Trimethylglycine</td></tr><tr><td>Magnesium Sulfate</td><td>Sigma-Aldrich</td><td>M3409-1ML</td><td>1 M</td></tr><tr><td>10x Bluejuice</td><td>Invitrogen</td><td>10816-015</td><td>gel loading buffer</td></tr><tr><td>SYBR green</td><td>Invitrogen</td><td>S7585</td><td>10,000x, visualize products in tubes</td></tr><tr><td>GelRed</td><td>Phenix Research Products</td><td>RGB-4103</td><td>10,000x, visualize products in gels</td></tr><tr><td>Lyophilized reagents</td><td>Gene Reach</td><td></td><td></td></tr><tr><td>Hydroxynaphthol blue</td><td>Fluka</td><td>33936-10G</td><td></td></tr><tr><td>ESEQuant tube scanner</td><td>Qiagen</td><td></td><td>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 the detection of the reaction products with HNB or fluorescent intercalating dye present in the reaction. 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. The inclusion of fluorescent intercalating dye in the reaction shows a strong fluorescence signal with UV light when 25, 50, and 100 copies of DNA templates are present. A tube scanner was used in this study for real-time detection. This machine can perform 8 reactions simultaneously. Figure 3 shows using the tube scanner to monitor the fluorescence signals in real-time with fluorescent intercalating dye presence. The fluorescence signals are above baseline after 14, 18, 21, and 23 min with 106, 104, 103, and 100 copies of DNA template in the reactions.
<img alt="Figure 1" src="/files/ftp_upload/53839/53839fig1.jpg" /> 
Figure 1. LAMP Results on Agarose Gel with Freshly Prepared Reagents (A) and Lyophilized Reagents (B). Reaction mixtures were incubated at 60 &#176;C for 60 min. (A) Lane 1, 100 bp ladder; Lane 2 - 3, 4 - 5, 6 - 7 and 8 - 9 are reactions with 2.5 x 103, 2.5 x 102, 2.5 x 10, and no copies of plasmid DNA. (B) Lane 10 - 11, 12 - 13, 14 - 15, and 16 - 17 are reactions with 2.5 x 103, 2.5 x 102, 2.5 x 10, and 0 copies of plasmid DNA. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53839/53839fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53839/53839fig2.jpg" /> 
Figure 2. Methods to Detect LAMP Products. HNB (A) or fluorescent intercalating dye (B) were used to detect LAMP products. Reactions were carried out at 60 &#176;C for 60 min. Lane 1 to 5, reactions with 0, 10, 25, 50, and 100 copies of plasmid. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53839/53839fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53839/53839fig3.jpg" /> 
Figure 3. Real-time Detection of LAMP Products. Reactions were carried out at 60 &#176;C for 60 min. Reactions with 106 (black), 104 (red), 103 (green), 100 (yellow), 10 (blue), 1 (orange) and 0 (brown and light brown) copies of plasmid. Three detections were performed independently. They were highly reproducible. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53839/53839fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

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Immunology and Infection

10.4K Views.  Naval Medical Research Center. We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

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

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

A Loop-mediated Isothermal Amplification LAMP Assay for Rapid Identification of Bemisia tabaci

This paper reports the protocol for a rapid identification assay for Bemisia tabaci based on loop-mediated isothermal amplification (LAMP) technology. The protocol requires minimal laboratory training and can, therefore, be implemented on-site at points of entry for plant imports such as seaports and airports.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many ...

Environment

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in vegetable production annually. Phytophthora capsici is soil-borne and a persistent problem in vegetable fields due to its long-lived survival structures (oospores and chlamydospores) that resist weathering and degradation. The main method of dispersal is through the production of zoospores, which are single-celled, flagellated spores that can swim through thin films of water present on surfaces or in water-filled soil pores and can accumulate in puddles and ponds. Therefore, irrigation ponds can be a source of the pathogen and initial points of disease outbreaks. Detection of P. capsici in irrigation water is difficult using traditional culture-based methods because other microorganisms present in the environment, such as Pythium spp., usually overgrow P. capsici making it undetectable. To determine the presence of P. capsici spores in water sources (irrigation water, runoff, etc.), we developed a hand pump-based filter paper (8-10 &#181;m) method that captures the pathogen&#8217;s spores (zoospores) and is later used to amplify the pathogen&#8217;s DNA through a novel loop-mediated isothermal amplification (LAMP) assay designed for the specific amplification of P. capsici. This method can amplify and detect DNA from a concentration as low as 1.2 x 102 zoospores/mL, which is 40 times more sensitive than conventional PCR. No cross-amplification was obtained when testing closely related species. LAMP was also performed using a colorimetric LAMP master mix dye, displaying results that could be read with the naked eye for on-site rapid detection. This protocol could be adapted to other pathogens that reside, accumulate, or are dispersed via contaminated irrigation systems.

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

We developed a method to detect Phytophthora capsici zoospores in water sources using a filter paper DNA extraction method coupled with a loop-mediated isothermal amplification (LAMP) assay that can be analyzed in the field or in the lab.

Phytophthora capsici is a devastating oomycete pathogen that affects many important solanaceous and cucurbit crops causing significant economic losses in ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Biology

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the absence of effective control clinical coccidiosis can be devastating. Resistance to the chemoprophylactics frequently used to control Eimeria is common and sub-clinical infection is widespread, influencing feed conversion ratios and susceptibility to other pathogens such as Clostridium perfringens. Despite the availability of polymerase chain reaction (PCR)-based tools, diagnosis of Eimeria infection still relies almost entirely on traditional approaches such as lesion scoring and oocyst morphology, but neither is straightforward. Limitations of the existing molecular tools include the requirement for specialist equipment and difficulties accessing DNA as template. In response a simple field DNA preparation protocol and a panel of species-specific loop-mediated isothermal amplification (LAMP) assays have been developed for the seven Eimeria recognised to infect the chicken. We now provide a detailed protocol describing the preparation of genomic DNA from intestinal tissue collected post-mortem, followed by setup and readout of the LAMP assays. Eimeria species-specific LAMP can be used to monitor parasite occurrence, assessing the efficacy of a farm&#8217;s anticoccidial strategy, and to diagnose sub-clinical infection or clinical disease with particular value when expert surveillance is unavailable.

Loop-mediated Isothermal Amplification LAMP Assays for the Species-specific Detection of Eimeria that Infect Chickens

Diagnosis of Eimeria infection in chickens remains demanding. Parasite morphology- and host pathology-led approaches are commonly inconclusive, while molecular approaches based on PCR have proven demanding in cost and expertise. The aim of this protocol is to establish loop-mediated isothermal amplification (LAMP) as a straightforward molecular diagnostic for eimerian infection.

Eimeria species parasites, protozoa which cause the enteric disease coccidiosis, pose a serious threat to the production and welfare of chickens. In the ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a replicative niche. A cohort of effectors are translocated through this system into the host cell to manipulate host processes and allow the establishment of a unique lysosome-derived vacuole for replication. The method presented here involves the combination of two well-established techniques: specific gene silencing using siRNA and measurement of effector translocation using a FRET-based substrate that relies on &#946;-lactamase activity. Applying these two approaches, we can begin to understand the role of host factors in bacterial secretion system function and effector translocation. In this study we examined the role of Rab5A and Rab7A, both important regulators of the endocytic trafficking pathway. We demonstrate that silencing the expression of either protein results in a decrease in effector translocation efficiency. These methods can be easily modified to examine other intracellular and extracellular pathogens that also utilize secretion systems. In this way, a global picture of host factors involved in bacterial effector translocation may be revealed.

Applying Fluorescence Resonance Energy Transfer FRET to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Investigating the interactions between bacterial pathogens and their hosts is an important area of biological research. Here, we describe the necessary techniques to measure effector translocation by Coxiella burnetii during siRNA gene silencing using BlaM substrate.

Coxiella burnetii, the causative agent of Q fever, is an intracellular pathogen that relies on a Type IV Dot/Icm Secretion System to establish a ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Bioengineering

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Human Respiratory Bacterial Infection Diagnosis Using Isothermal Amplification

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will provide practical guidelines for managing and treating RTIs. This study describes a method for rapidly detecting bacterial pathogens that cause respiratory tract infections via multi-channel loop-mediated isothermal amplification (LAMP). LAMP is a sensitive and specific diagnostic tool that rapidly detects bacterial nucleic acids with high accuracy and reliability. The proposed method offers a significant advantage over traditional bacterial culturing methods, which are time-consuming and often require greater sensitivity for detecting low levels of bacterial nucleic acids. This article presents representative results of K. pneumoniae infection and its multiple co-infections using LAMP to detect samples (sputum, bronchial lavage fluid, and alveolar lavage fluid) from the lower respiratory tract. In summary, the multi-channel LAMP method provides a rapid and efficient means of identifying single and multiple bacterial pathogens in clinical samples, which can help prevent the spread of bacterial pathogens and aid in the appropriate treatment of RTIs.

Author Spotlight: Advancing Rapid Detection of Respiratory Pathogens Using Microfluidic Chip 

Various bacterial pathogens can cause respiratory tract infections and lead to serious health issues if not detected accurately and treated promptly. Rapid and accurate detection of these pathogens via&#160;loop-mediated isothermal amplification provides effective management and control of respiratory tract infections in clinical settings.

Respiratory tract infections (RTIs) are among the most common problems in clinical settings. Rapid and accurate identification of bacterial pathogens will ...

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 &#176;C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration&#8217;s Bacteriological Analytical Manual (BAM) Chapter 5.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation

Loop-mediated isothermal amplification (LAMP) is an isothermal nucleic acid amplification test (iNAAT) that has attracted broad interest in the pathogen detection field. Here, we present a multi-laboratory-validated Salmonella LAMP protocol as a rapid, reliable, and robust method for screening Salmonella in animal food and confirming presumptive Salmonella from culture isolation.

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, ...

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

Summary

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Chapters in this video

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci

Detection of Phytophthora capsici in Irrigation Water using Loop-Mediated Isothermal Amplification

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Loop-mediated Isothermal Amplification (LAMP) Assays for the Species-specific Detection of Eimeria that Infect Chickens

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Applying Fluorescence Resonance Energy Transfer (FRET) to Examine Effector Translocation Efficiency by Coxiella burnetii during siRNA Silencing

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Optimized PCR-based Detection of Mycoplasma

Rapid Detection of Bacterial Pathogens Causing Lower Respiratory Tract Infections via Microfluidic-Chip-Based Loop-Mediated Isothermal Amplification

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation