Over the past years, molecular tests were developed to allow a clear and rapid species diagnosis when external morphological characters are not fully discriminant. Most of these tests are based on the use of species-specific primers, which relies on the definition of PCR primer sets that amplify the DNA belonging to only one species.
Given the importance of the genus Bandicota as a vector for several human diseases, CERoPath project developed such a molecular test to unambiguously assign Bandicota specimens to a single species. This method which could be extended to most other species has the advantages of:
- being cheap and suitable for investigations of large series of specimens;
- being useful for field surveys, since it requires simple and easily accessible DNA samples, which are easy to conserve in field trip conditions (ethanol stored at room temperature);
- being also applicable on dry and/or osteological material, allowing a precise species assignment for specimens in collections and museums.
The test is conducted in two steps. The first step allows discrimination of Bandicota DNA samples from other samples belonging to other genera (PCR1 ). The specificity of the test was verified using the 27 different rodent species sampled during the CERoPath project. The second step allows the two different Bandicota species inhabiting the study area to be distinguised (PCR2 ). It is possible to combine these two steps as a multiplex PCR .
Table 3 : Primers used to discriminate Bandicota indica from Bandicota savilei and from other rodent species.
Gene | Type of primer | Name | Primer |
Cyt b gene | generalist | L14723 | 5’ACC-AAT-GAC-ATG-AAA-AAT-CAT-CGT-T 3’ |
generalist | H15915 | 5’ CT-CCA-TTT-CTG-GTT-TAC-AAG-AC 3’ | |
specific of Bandicota genera | FBan | 5’ TTA-ATC-TTA-GCC-TTC-ATA-CCT-CTA 3’ | |
Dloop gene | generalist | F1043 | 5’ GGC-CAA-CTA-GCA-TCC-ATC-AG 3’ |
generalist | R1594 | 5’ GAC-GGC-TAT-GTT-GAG-GAA-GG 3’ | |
specific of Bandicota savilei | FBsa | 5’ CAT-ACA-CCA-TAA-AGT-CCA-AAA-TCA 3’ |
Protocol: PCR-test for Bandicota species assignment
1- Perform PCR amplifications in 10 µL reaction volumes with 2 µL of DNA matrix pre-diluted forty times after extraction (if using Qiagen Spin-Column Protocol). 2- Add 0.2 µL for the FBan primers and 0.2 µL for the other primer pairs L14723 and H15915 (10 µM) (Table 3), 0.4 µL of a Deoxynucleoside-triphosphate mixture (dNTP 2.5 mM), 1µl of reaction red Coral Load buffer (10*) (Qiagen), 0.5 µL of MgCl2 (25 mM) and 0.1 µL of Taq DNA polymerase (Qiagen). 3- Use the following thermal cycling parameters: 2 min at 94°C, 30 cycles (30 s at 94°C, 30 s at 52°C, 60 s at 72°C), with a final extension of 10 min at 72°C 4- Visualize band products by migrating the DNA fragments in an agarose gel to 2.5% so as to clearly distinguish the specific PCR-fragments. Two PCR-fragments, one of 320 bp (FBan, H15915) and a second one of 1,150 bp (L14723, H15915) are obtained for Bandicota samples when only one PCR-fragment of 1,150 bp (L14723, H15915) is obtained for samples belonging to other genus (Fig. 47).
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Figure 47 : PCR1 agarose gel to differentiate genus Rattus and genus Bandicota (Photo: Chaval Y.) |
1- Perform PCR amplifications in 10µl reaction volumes with 2 µL of DNA matrix pre-diluted forty times after extraction (if using Qiagen Spin-Column Protocol). 2- Add 0.2 µL for the FBsa primers and 0,2µl for the other primer pairs F1043 and R15945 (10 µM) (Table 3), 0.4 µL of a Deoxynucleoside-triphosphate mixture (dNTP 2.5 mM), 1 µL of reaction red Coral Load buffer (10*) (Qiagen), 0.5 µL of MgCl2 (25 mM) and 0.1 µL of Taq DNA polymerase (Qiagen). 3- Use the following thermal cycling parameters: 2 min at 94°C, 30 cycles (30 s at 94°C, 30 s at 52°C, 60 s at 72°C), with a final extension of 10 min at 72°C. 4- Visualize band products by migrating the DNA fragments in an agarose gel to 2.5%. Two PCR-fragments, one of 350 bp (FBsa, R1594) and a second one of 750 bp (F1043, R1594) are obtained for B. savilei while only one of 750 bp (F1043, R1594) is obtained for B. indica and samples of Rattus genus (Fig. 48).
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Figure
48
: PCR2 agarose gel to differentiate
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Further tests revealed that mixing these primers (FBan, L14723, H15915, FBsa, F1043, R1594) within a single PCR produces reliable results.
1- Perform PCR amplifications in 10 µL reaction volumes with 2 µL of DNA matrix pre-diluted forty times after extraction. 2- After adjusting the PCR reaction’s conditions, each specific reaction includes 0.2 µL for all the six primers (10 µM) (Table 3), 0,4µl of a Deoxynucleoside-triphosphate mixture (dNTP 2.5 mM), 1µl of reaction red Coral Load buffer (10*) (Qiagen), 0.2 µL of MgCl2 (25 mM) and 0.1 µL of Taq DNA polymerase (Qiagen). 3- Use the following thermal cycling parameters: 2 min at 94°C, 30 cycles (30 s at 94°C, 30 s at 52°C, 60 s at 72°C), with a final extension of 10 min at 72°C. 4- Visualize band products by migrating the DNA fragments in an agarose gel to 2.5%. Five PCR-fragments (320 bp (FBan, H15915), 350 bp (FBsa, R1594), 750 bp (F1043, R1594), 950 bp (FBan, R1594) and 1,150 bp (L14723 ,H15915)) are obtained for B. savilei. Four PCR-fragments (320 bp (FBan, H15915), 750 bp (F1043, R1594), 950 bp (FBan, R1594), 1,150 bp (L14723, H15915)) are obtained for B. indica. Two PCR-fragments (750 bp (F1043, R1594) and 1150 (L14723, H15915)) are obtained for Rattus genus (Fig. 49).
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Figure 49 : Multiplex PCR agarose gel to differentiate the genus Bandicota and R. tanezumi. (Photo: Chaval Y.) |
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