Standardization of Multiplex Real-Time PCR for the diagnosis of Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) in Felis silvestris catus


  • Claudia Velilla Universidad CES
  • Juliana Martínez Universidad CES
  • Juliana Martínez Universidad CES
  • María Soledad González Universidad CES
  • María Soledad González Universidad CES

Palabras clave:

Molecular diagnosis, Feline Leukemia virus, Multiplex PCR, Feline Immunodeficiency virus.


Diseases such as Feline Immunodeficiency Virus (VIF) and Feline Leukemia Virus (VLFe) are highly prevalent in domestic cats, due to their ease of transmission. Clinical signs are similar to other infections causing difficulties in diagnosis, therefore, they should be analyzed by laboratory tests; Currently available tests have some disadvantages, a multiplex real-time PCR was standardized with Taqman probes, designed for detection of infection with VIF, VLFe or both. Sensitivity (0.53 / 0.26) and specificity (0.46 / 0.74) for VLFe and VIF respectively, were calculated with Broemeling’s methodology, which considers that the reference test against which it is compared is not a Gold standard, according to these results it is necessary to increase the sample size; However, multiplex PCR is a very sensitive methodology, it was validated In silico form and presented congruent results when performed in vivo. Estandarización de PCR múltiple en tiempo real para el diagnóstico de sida y leucemia en Felis silvestris catusLas enfermedades producidas por virus como sida y leucemia son altamente prevalentes en felinos domésticos, debido a su facilidad de transmisión, presentan signos clínicos similares a otras infecciones que pueden generar dificultades en el diagnóstico, por lo tanto, se deben analizar mediante pruebas de laboratorio; los exámenes disponibles en la actualidad presentan algunas desventajas, por ello se estandarizó una PCR múltiple en tiempo real con sondas Taqman que fue diseñada para detección de infección con el virus de inmunodeficiencia felina (VIF), el virus de leucemia felina (VLFe) o mixtas. Se calculó la sensibilidad (0,53/0,26) y la especificidad (0,46/0,74) para leucemia y sida respectivamente con la metodología de Broemeling que considera que la prueba de referencia contra la cual se compara no es un referente verdadero (Gold standard), según estos resultados se concluye que es necesario aumentar el tamaño de muestra; sin embargo, la PCR múltiple es una metodología muy sensible, fue validada de forma in silico y presentó resultados congruentes al realizarla In vivo.Palabras clave: Diagnóstico molecular, leucemia felina, PCR múltiple, sida felino. Padronização da PCR múltipla em tempo real para o diagnóstico de AIDS e leucemia em Felis silvestris catusDoenças causadas por vírus como AIDS e leucemia são altamente prevalentes em gatos domésticos, devido a sua facilidade de transmissão com sinais clínicos semelhantes a outras infecções que podem causar dificuldades no diagnóstico, portanto devem ser analisados por exames laboratoriais; Os testes atualmente disponíveis têm algumas desvantagens, portanto uma PCR em tempo real múltipla foi padronizada com sondas Taqman para a detecção de infecção pelo vírus da imunodeficiência felina (FIV), vírus da leucemia felina (VLFe) ou misto . A sensibilidade (0,53/0,26) e a especificidade (0,46 / 0,74) para leucemia e AIDS foram calculadas, respectivamente, com a metodologia de Broemeling, que considera que o teste de referência com o qual é comparado não é um referência verdadeira (teste de ouro), de acordo com esses resultados, conclui-se que é necessário aumentar o tamanho da amostra, entretanto, a PCR múltipla é uma metodologia muito sensível, foi validada na forma silico e apresentou resultados congruentes quando realizada in vivo.Palavras-chave: Diagnóstico molecular, leucemia felina, PCR múltiplo, AIDS felina.


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Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. “Basic local alignment

search tool.” J. Mol. Biol. 1900; 215: 403-410.

Arjona A., Barquero N., Domenech A., Tejerizo G., Collado V., et al. Evaluation of

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

feline immunodeficiency virus (FIV). J Feline Med Surg. 2007; 9 (1): 14-22.

Arjona, A., Escolar, E., Soto, I., Barquero N., Martin, D., et al. Seroepidemiological Survey

of Infection by Feline Leukemia Virus and Immunodeficiency Virus in Madrid and

Correlation with Some Clinical Aspects. J Clin Microbiol. 2000; 38 (9): 3448-3449.

Broemeling, L. D, Advanced Bayesian methods for medical test accuracy. Diagnostics

(Basel). 2011 Dec; 1 (1): 1-35.

Bryan Kestenbaum. Epidemiology and Biostatistics. New York, NY: Springer New

York; 2009.

Dong J., Olano J. P., McBride J. W., Walker D. H. Emerging Pathogens: Challenges

and Successes of Molecular Diagnostics. J Mol Diagn. 2008; 10 (3): 185-197.

Gautam R, Mijatovic-Rustempasic S, Esona MD, Tam KI, Quaye O, Bowen MD.

One-step multiplex real-time RT-PCR assay for detecting and genotyping wildtype

group A rotavirus strains and vaccine strains (Rotarix® and RotaTeq®) in

stool samples. PeerJ. 2016; 4:e1560.

Greggs, W. M., Clouser, C. L., Patterson, S. E., & Mansky, L. M. Discovery of drugs that

possess activity against feline leukemia virus. J Gen Virol. 2012; 93 (4): 900-905.

Hernández C, Cucunubá Z, Flórez C, Olivera M, Valencia C, et al. Molecular Diagnosis

of Chagas Disease in Colombia: Parasitic Loads and Discrete Typing Units

in Patients from Acute and Chronic Phases. PLoS Negl Trop Dis 10 (9): e0004997.

Hong Naa, Willem Huismanb, Kristofor K., Ellestada Tom R. Phillips, Christopher

Powerad. Domain -and nucleotide- specific Rev response element regulation of

feline immunodeficiency virus production. Virology, 2010; 404 (2): 246-260.

IDEXX Laboratories, “Feline Leukemia Virus Antigen-Feline Immunodeficiency

Virus Antibody Test Kit”, Test insert, Westbrook, Maine 2014; https://www.idexx.


J. Hoorfar, B. Malorny, A. Abdulmawjood, N. Cook, M. Wagner et al. Practical Considerations

in Design of Internal Amplification Controls for Diagnostic PCR Assays. J

Clin Microbiol, 2004; 42 (5): 1863-1868.

Kent WJ, Sugnet CW, Furey TS, Roskin KM, Pringle TH, Zahler AM, Haussler D. The

human genome browser at UCSC. Genome Res. 2002; 12 (6): 996-1006

Kenyon, J. C., Lever A. M. L. The Molecular Biology of Feline Immunodeficiency

Virus (FIV). Viruses 2011; 3 (12): 2192-2213.

Kim WS, Chong CK, Kim HY, Lee GC, Jeong W, et al. Development and clinical

evaluation of a rapid diagnostic kit for feline leukemia virus infection. J Vet Sci,

; 15 (1): 91-97.

Little, S., Bienzle, D., Carioto, L., Chisholm, H., O’Brien, E. et al. Feline leukemia virus

and feline immunodeficiency virus in Canada: Recommendations for testing and

management. Can Vet J., 2011; 52 (8): 849-855.

Little, S., Sears, W., Lachtara, J., Bienzle D., Seroprevalence of feline leukemia

virus and feline immunodeficiency virus infection among cats in Canada. Can Vet

J., 2009; 50 (6): 644-648.

Mathevon Y, Foucras G, Falguières R, Corbiere F. Estimation of the sensitivity and

specificity of two serum ELISAs and one fecal qPCR for diagnosis of paratuberculosis

in sub-clinically infected young-adult French sheep using latent class

Bayesian modeling. BMC Vet Res. 2017; 13 (1) 230.

Beall MJ, Buch J, Cahill RJ, Clark G, Hanscom J, Estrada M, et al. Evaluation of a

quantitative enzyme-linked immunosorbent assay for feline leukemia virus p27

antigen and comparison to proviral DNA loads by real-time polymerase chain

reaction. Comp Immunol Microbiol Infect Dis. 2019; 67: 101348.

Mohammadi H., Bienzle D. Pharmacological Inhibition of Feline Immunodeficiency

Virus (FIV). Viruses, 2012; 4 (12): 708-724.

Munro H. J., Berghuis L., Lang A. S., Rogers L., Whitney H. Seroprevalence of feline

immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in shelter cats on

the island of Newfoundland, Canada. Can J Vet Res., 2014; 78 (2): 140-144.

Murphy B., Vapniarsky N., Hillman C., Castillo D., McDonnel S., Moore P., Sparger

E. E., et al. FIV establishes a latent infection in feline peripheral blood CD4+ T

lymphocytes in vivo during the asymptomatic phase of infection. Retrovirology,

; 9 (1): 12.

Nunes MS, T Heuer C, Marshall J, Sanchez J, Thornton R, et al. epiR: Tools for

the Analysis of Epidemiological Data. 2015 [cited 2015 Nov 20]. URL: https://

O_UZO_LU1 T., MUZ D. Prevalences of Feline Coronavirus (FCoV), Feline Leukaemia

Virus (FeLV), Feline Immunodeficiency Virus (FIV) and Feline Parvovirus

(FPV) among domestic cats in Ankara, Turkey. Med Vet. 2013; 164 (11): 511-516.

Perhari_ M., Bi_in M., Stare_ina V., Milas Z., Turk N., et al. Phylogenetic characterization

of feline immunodeficiency virus in naturally infected cats in Croatia indicates

additional heterogeneity of subtype B in Europe. Arch Virol. 2016; 161(9):


PrimerQuest® program, IDT, Coralville, USA. Retrieved 12 December, 2012. Kent WJ, Sugnet CW, Furey TS, Roskin KM,

Pringle TH, Zahler AM, Haussler D. The human genome browser at UCSC. Genome

Res. 2002 Jun;12 (6): 996-1006.

Qurollo B. A., Riggins D., Comyn A., Zewde M. T., Breitschwerdt E. B. Development

and Validation of a Sensitive and Specific sodB-Based Quantitative PCR Assay for

Molecular Detection of Ehrlichia Species. J Clin Microbiol. 2014; 52 (11): 4030-4032.

R Core Team, R: A language and environment for statistical computing. R Foundation

for Statistical Computing, Vienna, Austria. 2015 [cited 2015 Nov 20]. URL:

Radstrom P, Lofstrom C, Lovenklev M, Knutsson R, Wolffs P. Strategies for Overcoming

PCR Inhibition. Cold Spring Harb Protoc. 2008; 2008(4): pdb.top20-pdb.


Rådström P, Knutsson R, Wolffs P, Lövenklev M, Löfström C. Pre-PCR Processing:

Strategies to Generate PCR-Compatible Samples. Mol Biotechnol. 2004;26(2):


Ramírez H, Autran M, García MM, Carmona MÁ, Rodríguez C, Martínez HA. Genotyping

of feline leukemia virus in Mexican housecats. Arch Virol. 2016; 161 (4):


Rohn, J., Linenberger M. L., Hoover E. A., Overbaugh J. Evolution of Feline Leukemia

Virus Variant Genomes with Insertions, Deletions, and Defective Envelope

Genes in Infected Cats with Tumors. J Virol 1994; 68: 2458-2467.

Sales M., Fonseca Junior, A. A., Orzil, L., Padilha Alencar, A., Silva, M. R., et al.

Validation of a real-time PCR assay for the molecular identification of Mycobacterium

tuberculosis, Braz J Microbiol. 2014; 45 (4): 1363-1369.

Sonntag O. Haemolysis as interference factor in clinical chemistry. J Clin Chem

Clin Biochem. 1986; 24: 127-39.

Tandon R, Cattori V, Willi B, Lutz H, Hofmann-Lehmann R. Quantification of endogenous

and exogenous feline leukemia virus sequences by real-time PCR assays. Vet

Immunol Immunopathol. 2008;123 (1-2):129-33.

Velilla Salim, Tique Vaneza, Sanchez Alba, Alvarez Leonardo, Rios Rodrigo. Seroprevalencia

del virus de leucemia e inmunodeficiencia felina en gatos de Montería,

Córdoba. Rev. Med. Vet. Zoot. 2009; 56: 85-94.

Westman ME, Malik R, Hall E, Sheehy PA, Norris JM. Comparison of three feline

leukaemia virus (FeLV) point-of-care antigen test kits using blood and saliva.

Comp Immunol Microbiol Infect Dis. 2017; 50: 88-96.

Westman ME, Malik R, Norris JM. Diagnosing feline immunodeficiency virus (FIV)

and feline leukaemia virus (FeLV) infection: an update for clinicians. Aust Vet J.

; 97 (3): 47-55.

Zhou, X., McClish, D. K., Obuchowski N. A, Statistical methods in diagnostic medicine

nd ed. Hoboken, N.J: Wiley; 2011.