Detección, discriminación y cuantificación de pequeñas secuencias de nucleótidos por Espectroscopía Infrarroja por Transformada de Fourier (FTIR) en la identificación molecular del Virus Del Papiloma Humano
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Fecha
2021
Autores
Guerra Simanca, Martha Isabel
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Ediciones Universidad Simón Bolívar
Facultad de Ciencias Básicas y Biomédicas
Facultad de Ciencias Básicas y Biomédicas
Resumen
En este trabajo investigativo se evaluó el uso de la técnica de espectroscopía infrarroja
con transformada Fourier por reflexión total atenuada (ATR-FTIR) en la detección,
discriminación y cuantificación de pequeñas secuencias de nucleótidos aplicado en la
identificación molecular de genotipos de virus de papiloma humano (VPH). En una
primera parte se demostró la viabilidad de la técnica ATR-FTIR en la diferenciación de
pequeñas secuencias de ADN de una sola cadena, para esto se generó un modelo de
regresión para la cuantificación del porcentaje de nucleótido (%N, para cada nucleótido
%A, %C, %T y %G) por el método multivariado de mínimos cuadrados parciales (PLS) y
se analizaron las señales espectrales por ATR-FTIR de estas secuencias. El error de los
modelos para la cuantificación del %N estuvo entre 0.9-1.2%.
En una segunda parte se implementó la técnica ATR-FTIR en la identificación de VPH.
Para esto se generaron modelos multivariados por el método de análisis discriminante
PLS (PLS-DA) para la predicción de los genotipos de VPH 16, 31, 35, 51 y 66. Esto se
realizó a partir de espectros de los productos de amplificados de ADN por la técnica de
reacción en cadena de la polimerasa en tiempo real (real-time PCR), los modelos fueron
diseñados usando los espectros ATR-FTIR de los controles positivos y negativos de los
productos de real-time PCR. Estos modelos fueron usados para predecir muestras
clínicas de seis mujeres y los resultados fueron contrastados con la técnica convencional
real-time PCR. Todas las muestras fueron predichas con el mismo genotipo de VPH
validado por real-time PCR.
In this research, the Fourier transform infrared spectroscopy by attenuated total reflection (ATR-FTIR) technique is used to detect, discriminate, and quantify small nucleotide sequences. This was applied in the molecular identification of genotypes of human papillomavirus (HPV). Initially, I demonstrated the viability of the ATR-FTIR technique in the differentiation of small single-stranded DNA sequences, for this, a model was generated to quantify the nucleotide percentage (% N, for each nucleotide % A, % C,% T, and % G) by the multivariate method of partial least squares (PLS) and the spectral signals of these sequences were analyzed by ATR-FTIR. The error of the models for the quantification of % N was between 0.9-1.2%. Finally, to identify HPV, I implemented the ATR-FTIR technique. For this, multivariate models were generated by the PLS discriminant analysis method (PLS-DA) in the prediction of HPV genotypes 16, 31, 35, 51 and 66. This was performed from spectra of DNA amplification products using the realtime polymerase chain reaction (real-time PCR) technique; the models were designed using the ATR-FTIR spectra of the positive and negative controls of the real-time PCR products. These models were used to predict clinical samples from six women, and the results were contrasted with the conventional real-time PCR technique. All samples were predicted with the same HPV serotype found by real-time PCR.
In this research, the Fourier transform infrared spectroscopy by attenuated total reflection (ATR-FTIR) technique is used to detect, discriminate, and quantify small nucleotide sequences. This was applied in the molecular identification of genotypes of human papillomavirus (HPV). Initially, I demonstrated the viability of the ATR-FTIR technique in the differentiation of small single-stranded DNA sequences, for this, a model was generated to quantify the nucleotide percentage (% N, for each nucleotide % A, % C,% T, and % G) by the multivariate method of partial least squares (PLS) and the spectral signals of these sequences were analyzed by ATR-FTIR. The error of the models for the quantification of % N was between 0.9-1.2%. Finally, to identify HPV, I implemented the ATR-FTIR technique. For this, multivariate models were generated by the PLS discriminant analysis method (PLS-DA) in the prediction of HPV genotypes 16, 31, 35, 51 and 66. This was performed from spectra of DNA amplification products using the realtime polymerase chain reaction (real-time PCR) technique; the models were designed using the ATR-FTIR spectra of the positive and negative controls of the real-time PCR products. These models were used to predict clinical samples from six women, and the results were contrasted with the conventional real-time PCR technique. All samples were predicted with the same HPV serotype found by real-time PCR.
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Palabras clave
ATR-FTIR, VPH, ADN, Nucleótidos, Cáncer cervicouterino, PLS, PLS-DA, real-time PCR, HPV, DNA, Nucleotides, Cervical cancer