Evaluation of a novel nested PCR for the routine diagnosis of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV)

Abstract

Laboratory diagnosis of feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) usually involves both viruses, as the clinical signs are similar and coinfection may occur. Serological methods may not represent an accurate diagnosis: maternal antibodies or cross-reactions may give false positive results to FIV, and false negative results may occur in latent FeLV status, or in certain FIV infection stages. A nested polymerase chain reaction (PCR) technique was designed to detect FeLV, FIV and feline endogenous retrovirus simultaneously. The detection of endogenous sequences was considered indicative of successful DNA extraction. The technique was used to diagnose FIV and FeLV in the blood cells of 179 cats. The kappa value with the serological data was 0.69 for FeLV and 0.87 for FIV. The joint detection of FeLV and FIV by this novel nested PCR is sensitive, specific, fast and convenient, and its applicability for clinical diagnosis is promising, as the direct evidence of the presence of the virus is more realistic than the indirect data provided by the serological detection.

Fig 1

Optimization of the number of cycles for the first and second PCR reactions. For the first PCR reaction, the number of cycles is read across and for the second it is read down. The positions of the bands of 1138 bp corresponding to FIV, 306 bp to FeLV and 257 bp to the feline endogenous retroviruses are shown. Lanes 1, 10⁵ Ho6 (FIV infected) cells; lanes 2, 10⁵ FL-74 (FeLV infected) cells; lanes 3, 10⁵ CRFK (feline endogenous retroviruses control) cells; lanes 4, 10⁷ U937 (negative control of DNA) cells; and lanes 5, double distilled water.

Fig 2

Optimization of the time of extension at 72°C for the first and second PCR reactions. For the first PCR reaction, the time is read across and for the second it is read down. The positions of the bands of 1138 bp corresponding to FIV, 306 bp to FeLV and 257 bp to the feline endogenous retroviruses are shown. Lanes 1, 10⁶ FL-74 (FeLV infected) cells; lanes 2, 10³ FL-74 cells; lanes 3, 10⁶ Ho6 (FIV infected) cells; lanes 4, 10³ Ho6 cells; lanes 5, 10⁶ CRFK (feline endogenous sequences control) cells; and lanes 6, double distilled water.

Fig 3

Assay of sensitivity. Samples were amplified using 35 cycles for the first PCR reaction and 25 for the second. Extension was performed at 72°C for 3 min in the first PCR reaction, and 90 s in the second PCR reaction. The position of the bands of 1138 bp corresponding to FIV, 306 bp to FeLV and 257 bp to the feline endogenous retroviruses is shown. Lane 1, 10⁶ FL-74 (FeLV infected) cells; lane 2, 10⁵ FL-74 cells; lane 3, 10⁴ FL-74 cells; lane 4, 10³ FL-74 cells; lane 5, 10² FL-74 cells; lane 6, 10 FL-74 cells; lane 7, one FL-74 cell; lane 8, FeLV DNA control; lane 9, CRFK (endogenous retroviruses control); lane 10, double distilled water; lane 11, 100 kb DNA ladder; lane 12, 10⁶ Ho6 (FIV infected) cells; lane 13, 10⁵ Ho6 cells; lane 14, 10⁴ Ho6 cells; lane 15, 10³ Ho6 cells; lane 16, 10² Ho6 cells; lane 17, 10 Ho6 cells; lane 18, 1 Ho6 cell; lane 19, FIV DNA control; and lane 20, double distilled water.