Standardization of Reverse Transcription Loop-Mediated Isothermal Amplification (Rt-Lamp) Diagnostic Test for Rapid Detection of Foot and Mouth Disease Virus

The foot and mouth disease virus (FMDV) is a member of aphthovirus, family picornavirdae (Grubman and Baxt, 2004). It has seven serotypes O, A, C, Asia-1, Sat-1, Sat-2 and Sat-3 (Alexandersen et al., 2003). A large number of subtypes involved within each serotype. Only O, A and Asia-1 are geographically important in Pakistan. The mortality rates are less than 5% but still considered one of the most important diseases of animals because it causes heavy economic losses. The disease spreads in susceptible animal’s at large population level.

Conventional diagnosis of disease is done by ELISA and cell culture (Reid et al., 2000). These are time consuming and require special equipments in lab.

There is a need for rapid and accurate diagnosis for foot and mouth disease virus. According to the principles of Lamp targeted RNA can be amplified in a hot water bath or heating block under isothermal conditions. A set of four primers were used in this study (Table 1). These loop primers were hybridized by the stem loops except for the loop which were hybridized by inner primers. In this way it reduced the amplification time. In this specific reaction the combination of AMV Reverse Transcriptase and Bst DNA polymerase stimulate the DNA amplification in the same tube. (Chen et al., 2009). Amplification and detection of gene can be completed in a single step.

Table 1: Details of oligonucleotide primers used in the current study of RT-LAMP amplification for FMDV.

These four primers can recognized 6 distinct regions. That is why it is much more specific. It is a cost effective assay because it does not require special reagents or sophisticated equipments. Only the color change indicated the positive reaction.

The LAMP was also been used to diagnose FMD virus in China (Chen et al., 2011).

In this study, initially we used reference strains of FMD viruses i.e Strain O, A and Asia-1, in order to check the sensitivity of the assay against these three strains.

In Pakistan, three serotypes of foot and mouth virus are prevalent. These are O, A and Asia-1. These FMD isolates were already confirmed by rRT PCR (Reid et al., 2002). These positive reference serotypes were maintained at Animal Health, NARC, Islamabad. Primarily we used these reference serotypes in order to develop the test. These three serotypes were separately grown in LFBK cells.

The RNA extraction was done by using RN easy Mini Kit [Qiagen] kit. The resultant RNA was eluted in 40ul of elution buffer.

This assay was performed in PCRtubes. Primarily three type of mixtures being prepared before performing LAMP PCR.

These are:

1. Primer Mixture: All primers were rehydrated in TE buffer at room temperature. These were stock solutions .The working solutions were prepared by dissolving 1:10 dilution in water.
2. Reaction Mixture: The reaction mixture was prepared by mixing pcr- buffer, MgSO4, d NTPs and nuclease –free water.
3. Enzyme Mixture: Two enzymes i.e, Bst DNA polymerase and AMV Reverse Transcriptase were mixed together in 1:5 ratio respectively.
4. Hydroxy Nephthiol Blue: A concentration of 3mM solution was used as dye in the assay.

Briefly, a 25ul reaction mixture contained, reaction mix. 12.5ul, primer mix.3ul, enzyme mix. 1ul, Hydroxy Naphthol Blue. 1.2ul, nuclease free water 5.3ul and 2ul RNA template.

All the tubes were kept in heat block at 65C for 60 min and then followed by 80 C for 10 min.

Gene amplification detection was analyzed by agarose gel electrophoresis. A 2% agarosegel was dissolved in tris borate EDTA buffer. In this experiment the positive reaction changed to blue color. Otherwise negative reaction remained violet in color.

This color change can be easily observed by naked eye. There is no need for sensitive equipments.

Accurate and rapid diagnosis of FMD is the need of time for effective control of the disease. Field diagnostic tests were required with high sensitivity and specificity.

In past we were using different molecular tests for the diagnosis. These tests required sophisticated scientific equipments and very expensive. On the other hand LAMP PCR is cheap and very easy to perform. This test only took only 60 minutes and results can be seen with naked eyes. The present study was conducted to standardize the LAMP assay against known positive strains of FMD (A, O, Asia 1).

These strains were diluted from -1 up to – 5 dilutions to check the sensitivity and specificity of lamp products. Results showed that LAMP was most sensitive for FMD strain O (positive reaction upto -4 dilution) than others strains A and Asia -1 (positive reaction upto -3 dilutions).

For designing Primers for RT-LAMP a highly conserved region of the 3D gene was chosen with a set of common primers was designed according to the 3D genes of the virus isolates. Two inner primers (forward inner primer (FIP) and backward inner primer (RIP), two outer primers (F3 and R3) and two loop primers (F loop and R loop) will be designed for RT-LAMP using the Primer Explorer version 4 software. The primers F3 and R3 will also be used for RT-PCR. These primers identified 8 distinct region of FMD RNA. This made the assay more sensitive and specific (Notomi et al., 2000). In the present study, 3D pol RNA was targeted which is highy conserved region (Reid et al., 2009).

Reaction was completed in 1 hr and 10 min at temperatures 600 C. It was also tested in hot water bath on same temperature. Results were same in both techniques. As the results can be observed with naked eyes so it did not require gel electrophoresis .The color change of the reaction itself resulted that samples was positive or negative.

RT LAMP assay was standardized for FMD viruses in the study. This test can be used for field diagnosis of FMD virus. The only limitation was extraction of RNA which was done in laboratory. This test is rapid easy to perform and cost effective.

Khalid Naeem concieved the idea of the study. Tahira Kamal, Saeed-Ul-Hassan Khan, Amir Bin Zahoor, Naeem Riaz, Siddra Tayyab Akhtar wrote the article. Ghulam Muhammad Ali provided technical input at every step and did overall management of the article.