Site Monitoring and Field Performance

3.2 Site Monitoring and Field Performance

Collected shrimp samples from different sampling sites were tested for the presence of WSSV. Shrimp samples from Bulacan, Bataan and General Santos City were subjected to laboratory WSSV screening using the standard LAMP assay and developed WSSV diagnostic kit (Fig. 3, 4 and 5). Field diagnosis was supported by subsequent laboratory LAMP and PCR screening (Figure 3, 4 and 5).

Using the LAMP assay, 93% of the P. monodon samples from Bulacan (REGION 3) were tested positive for WSSV infection (Figure 3). The characteristic laddering pattern and the presence of smear are indicators of a positive LAMP reaction (Notomi et al ., 2000). Collected P. vannamei samples from Bataan (REGION 3) (Figure 4) and General Santos City (REGION 12) (Figure 5) harboured WSSV infection with a total of 80% and 73% of the samples, respectively.

Field testing of the LAMP based diagnostic kit in Davao showed no presence of WSSV infection. However, laboratory test using PCR revealed than 20% of the samples were positive(Fig. 6A) whereas the developed LAMP assay diagnosed 50% of the samples were positive (Fig. 6B).

Figure 6. Bulacan area Penaeusmonodon screening for WSSV using LAMP assay. (M)-DNA ladder, Well 1 – negative for WSSV, Wells 2-15 – positive for WSSV, (N)- Negative control

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

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Figure 7. Bataan area Penaeusvannamei screening for WSSV using LAMP assay. (M)-DNA ladder, Well 1, 12 and 13 – negative for WSSV, Wells 2-11, 14 and 15 – positive for WSSV, (N)- negative control

M 9 10 11 12 13 14 15 N

Figure 8. General Santos city area Penaeusvannamei screening for WSSV using LAMP assay. (M)-DNA ladder, Well 1, 9, 13 and 14 – negative for WSSV, Wells 2-8, 10-12 and 15 – positive for WSSV, (N)- negative control

Sept em ber 8 th – th 9 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

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Figure 9.Davao city area Penaeusvannamei screening for WSSV using PCR (A) and LAMP (B) assay. (M)-DNA ladder, (N)- negative control, samples 1-8 negative for WSSV via PCR (A) and LAMP (B), samples 13,14, 16 and 20 positive for WSSV via PCR (A), samples 9, 10, 12-15, 17-20 positive for WSSV via LAMP (B) assay

These results showed that the greater analytical sensitivity of the LAMP assay over PCR (Figure 1) translates to its diagnostic performance, testing a larger percentage of the samples positive for WSSV in comparison to PCR. In addition, sample number

20 which tested positive via PCR and LAMP but the DNA source of the sample came from a non-shrimp species. The work of Hossain et al ., (2004) and Flegel et al ., (2006) supported the field test finding stating that various crustaceans including crabs can carry infection. Additionally, similar studies on crabs have also shown their role as carriers of WSSV infection contributing to the spread of disease (Corbel et. al ., 2001; Flegel, 2001 and Huang et al ., 2001).

Diagnostic kit testing in Cebu (Region 7) showed WSSV positive results for samples 1-4. Laboratory screening confirmed that 25% and 63% of the samples were positive for WSSV via PCR and LAMP assay, respectively (Figure 7). The high sensitivity of the developed LAMP assay allows it to detect WSSV even in low copy numbers often undetectable by PCR.

The most recent disease surveillance in Bataan (Region 3 - Central Luzon) revealed samples positive for WSSV via the on-field LAMP assay 90% (Figure 8-C). Laboratory confirmation of the results have shown that 0% (0/10) and 60% of the sample tested were positive for WSSV via conventional PCR and nested PCR respectively (Figure 9-A and 9-B). The diagnostic performance of the LAMP assay was comparatively more effective than PCR and nested PCR. The utilization of the

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

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Figure 10. Cebu area Penaeusvannamei screening for WSSV using PCR (A) and

LAMP (B) assay. (M)-DNA ladder, (P) – positive control, (N)- negative control, samples 4,6,10,13 positive for WSSV via PCR (A), samples 1-7, 10, 13,14 positive for WSSV via LAMP (B) assay.

Sept em ber 8 th – 9 th 2015, Facult y of Biot echnology – Universit as At m a Jaya Yogyakart a

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Figure 11. Bataan area Penaeusmonodon screening for WSSV using conventional PCR (A), LAMP (B) assay and nested PCR (C). (M)-DNA ladder, (P)- positive control, (N)- negative control, samples 1-10 diagnosed negative for WSSV via conventional PCR (A), samples 2-10 diagnosed positive for WSSV via