Philippine Council for Agriculture, Aquatic, Forestry and Natural ResourcesResearch and Development (PCAARRD)Telephone: (63) (049) 536-0014 to 536-0015Fax: (63) (049) 536-0016/536-7922E-Mail: pcarrd.dost.gov.phWebsite: http://www.pcarrd.dost.gov.ph

Central Luzon State University (CLSU)Science City of Muñoz, Nueva EcijaTelephone: (107) 4560107Fax: 456-5202/456-0866Website: www.clsu.edu.ph

College of Veterinary Science and Medicine (CVSM)Central Luzon State University (CLSU)Telfax: 044-456-5877Website: clsucvsm.edu.ph

Ramon Magsaysay Center for Agricultural Resources and Environment Studies (RM-CARES)Email: clsu_rmcares@yahoo.comTelfax: 044-4560-704

Department of Agriculture (DA)Website:www.da.gov.phTelefax: (045) 961-3336 (045) 961-3472

About Andali PED RT-LAMP Test Kit

Importance of PED Surveillance

The ANDALI PED RT-LAMP test kit was created and introduced to the public because of the outbreak of Porcine Epidemic Diarrhea (PED) which occurred in the province of Batangas, killing 67.0% (11,414 head) of 17,115 sick pigs last May-August 2010. The economic losses due to this outbreak amounted to Php 9.131M. The ANDALI PED RT-LAMP test is a rapid diagnostic test that can produce fast and efficient results in less than an hour. Earlier detection and appropriate response to prevent the spread of the disease can reduce the total number of loss during the outbreak. Assuming that 50.0% of the 11,414 pigs affected by PED survived due to early detection, economic losses could have been reduced by Php 4.56M.

• Identify suspect PED carriers among breeder sows or any age of pigs that show slight diarrhea but no occurrence of PED outbreak yet.

• Diagnose PED among full blown severe diarrhea among sucklings.

• Implement early prevention and control strategies upon identifying suspect carriers and locations with impending PED outbreak.

Important Instructions • Heat block must always be preheated at desired

temperature (65oC) before actual use.

• Always run the test samples with positive and negative controls.

• Once opened, entire kit must be utilized.

• Tubes containing LAMP premixes and Reagent 3 with LAMP Dye must be stored in the freezer (-15oC).

• Unused LAMP premixes stored in the freezer expire after two months.

• The test must be conducted only in a dedicated room, not exposed to wind or moving air current to reduce contamination.

FOR MORE INFORMATION ,VISIT OR CONTACT:

Dr. Clarissa Yvonne J. DomingoCollege of Veterinary Science and Medicine (CVSM)

CENTRAL LUZON STATE UNIVERSITYScience City of MUñoz Nueva Ecija

Telfax: 044-456-5877E-mail Address: cyjd1793@clsucvsm.edu.ph

Website:clsucvsm.edu.ph

What is LAMP? “LAMP” stands for Loop-mediated Isothermal Amplification. This technology was developed by Notomi et al. (2000). It is a very sensitive, easy and time efficient method. The LAMP reaction proceeds at a constant temperature using only a water bath or heat block for a strand displacement and annealing reaction.

The LAMP is an established amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. This technology has been developed into commercially available detection kits for a variety of pathogens including bacteria and viruses. The primary objective of LAMP methodology is as a diagnostic tool to be employed in resource-limited laboratories in developing countries, where many fatal tropical diseases are endemic.

Andali PED RT-LAMP Test ProcedureGet tissue grinder, dip

tip in fecal sample

Tissuegrinder

Transfer sample into the whitetube containing Reagent 1. Mix

well with tissue grinder

Get syringe, takea few amount &

put 3 drops into theblue tube containing

Reagent 2.

Dip end of yellowplastic tip and

transfer intosmaller tube with

LAMP premix.

Close tube lid,shake gently andplace into well ofthe heat block pre-heated to 65oC.Wait for 30minutes.

Then removethe LAMP tube. Dip end of white plastic

tip into tube with LAMPDye & transfer intoLAMP tube.

Green color ispositive LAMPreaction.

Comparison of LAMP with other High End Diagnostic Tests

General Advantages of LAMP Assay over other Current Diagnostic Tests

• Portable (designed for field testing using a heating block or heat bath) and easy to use. Does not require high-tech apparatus and/or complex preparation from highly trained personnel (specific and processed samples)

• Rapid results (within the day)

• 97% diagnostic sensitivity (better than PCR)

• Excellent analytical specificity

• Robust ( works even in crudely extracted clinical samples)

Andali PED RT-LAMP based Diagnostic Test Kit

Uses and Benefits• LAMP has the potential to be used as a simple

screening assay in the field, diagnosis for clinicians and surveillance to predict possible future outbreaks.

• LAMP is isothermal which eradicates the need for expensive thermal cyclers used in PCR; process is applicable to other infectious animal disease diagnosis.

• LAMP is a novel nucleic acid amplification technique which, due to its simplicity, robustness and low cost, provide major advantages to all stakeholders.

• The amount of nucleic acid amplified in LAMP is considerably higher than PCR based amplification.

• LAMP is widely being studied for detecting infectious diseases caused by other viruses, bacteria, protozoa, fungi, yeasts, filaria and other helminths.

• LAMP has been observed to be less sensitive than PCR to inhibitors in complex samples such as blood, most likely due to the use of a different polymerase enzyme.

• This feature of LAMP may be useful in laboratories with limited resources or field settings where use of commercial kits for DNA or RNA extraction prior to LAMP reaction may be impractical.

Process of Collecting Fecal Samples

Materials Needed in Fecal Sample Collection• Sterile toothpick or

cotton swab• Clean, small plastic bag

with zip lock

• Disposable gloves• Cooler with coolants or

styro box with ice

Heat Block Heat Block is an equipment used for incubating sample for nucleic acid amplification.

Illuminator Illuminator is an equipment used for visualizing fluorescence in the LAMP Fluorescence test.

Gel Electrophoresis Gel Electrophoresis is an equipment used for electrophoresis.

Gel Documentation Apparatus Gel Documentation apparatus is the machine which visualizes the bands in the gel after electrophoresis and saves the results for documentation purposes.

Lamp Detection

How to Detect LAMP Results

Equipment Needed for LAMP

Other Detection Method Done in a Molecular Laboratory

In a LAMP assay, the reaction takes place in a single tube containing buffer, polymerase and primers. The tube is incubated at 65°C in a regular laboratory water bath or heat block that must maintain this temperature constantly for 30 minutes. The amplified product can be detected by the naked eye upon addition of the LAMP dye into the tube, turning the color of the dye from orange to green and by emitting a greenish-white fluorescence under the trans-illuminator.

Dye Test A visual color change from orange to green in the LAMP product indicates a positive result.

Gel Electrophoresis If an agarose gel electrophoresis of the LAMP product is performed, multiple bands of various sizes will be visualized at regular intervals. • Lanes with multiple bands – positive • Lanes with no bands – negative

Fluorescence Test Tubes showing white to greenish luminescence are positive after viewing under a UV-illuminator.