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Application of COI-LAMP for Detection of Dirofilaria immitis with High Sensitivity and Specificity in Epidemiological
Melih Gazi Genc1, Ufuk Erol2, Omer Faruk Sahın3
1Department of Veterinary Parasitology, Institute of Health Sciences, Sivas Cumhuriyet University, Sivas, Turkey.
Purpose:
Dirofilaria immitis, a mosquito-borne zoonotic nematode, has worldwide distribution and causes infections in domestic and wild animals. Microscopic, serological, and molecular diagnostic methods are used to investigate this parasite in the hosts. Molecular diagnostic methods are outstanding for their sensitivity and specificity. The LAMP method, which has been used in detecting many parasites with its high specificity and sensitivity in recent years, is also advantageous with its simplicity of application. This study aimed to use the COI-LAMP method in the diagnosis of D. immitis in different host species.
Methods:
LAMP primers specific for the COI gene of D. immitis were designed, and the method was optimized. Additionally, the sensitivity, specificity, and limit of detection of the LAMP method were determined, and the results were compared with those of the PCR method. Moreover, to demonstrate the effectiveness of the LAMP method in epidemiologic studies, 600 blood samples were collected from dogs (n:300) and cats (n:300) in different parts of Türkiye. gDNA obtained from these samples were researched with LAMP and PCR assays, and the results were compered. Level of agreement between assays was calculated with Cohen's kappa test.
Results:
The limit of detection of the LAMP method was determined to be 0.0048 ng/μL, while that of the PCR method was 0.48 ng/μL, indicating that the LAMP method was approximately 100 times more sensitive than PCR. The blood samples were examined in terms of D. immitis, and ten samples (1.66%) were found to be positive. In contrast, six samples (1%) were positive by PCR. D. immitis was detected in nine (3%) dogs and one (0.33%) cat by LAMP method, and this parasite was detected in six (2%) dogs by PCR. Dirofilaria immitis was not detected by PCR in cat samples. The kappa value was calculated as κ = 0.76; this result revealed that the "substantial" agreement between assays.
Conclusion:
Our results showed that COI-LAMP has high sensitivity in the diagnosis of D. immitis in different hosts. It was also understood that its use in epidemiological studies would be useful. Since it is critical to know more accurate epidemiological data in the fight against the disease, it will be useful to use more sensitive diagnostic methods, like LAMP, in studies to be conducted in this field.
