The production of efficient and safe vaccines against the infectious laryngotracheitis virus (ILTV) represents an
important biological process for the health of millions of poultry birds. In this context, the validation of quantitative
methodologies is important to guarantee that the results show the actual state of vaccines, allowing an adequate
dosage. In this study, we developed, standardized and validated a methodology following Good Laboratory Practices
(GLP) and the guidelines of the Food and Drug Administration (FDA) to evaluate and measure the titer of vaccines
produced in allantoic fluid (AF). Therefore, a real-time PCR (qPCR) methodology was designed for the detection and
quantification of the g... More
The production of efficient and safe vaccines against the infectious laryngotracheitis virus (ILTV) represents an
important biological process for the health of millions of poultry birds. In this context, the validation of quantitative
methodologies is important to guarantee that the results show the actual state of vaccines, allowing an adequate
dosage. In this study, we developed, standardized and validated a methodology following Good Laboratory Practices
(GLP) and the guidelines of the Food and Drug Administration (FDA) to evaluate and measure the titer of vaccines
produced in allantoic fluid (AF). Therefore, a real-time PCR (qPCR) methodology was designed for the detection and
quantification of the glycoprotein B gene (gB) of ILTV using SYBR Green I. For this purpose, an internal reference
material for ILTV (IRM-ILTV), along with a plasmid with part of the gene encoding the gB protein of ILTV and lacking
the rest of the ILTV genome were produced. The validation criteria showed that the qPCR assay has a Limit of
Detection (LoD) of 1.017 × 105
genome copies/µL of AF which guarantees high precision, and a Limit of
Quantification (LoQ) of 3.39 × 105 genome copies/µL of AF. The confidence limit for confirming the presence of ILTV
with a conventional specific PCR obtained a LoD of 2.034 × 103
genome copies/µL of AF. These parameters
demonstrated the safety and accuracy of the correct detection and quantification of the ILTV viral load in vaccines
produced in AF. Hence, this procedure constitutes an important complementary tool for the quality assurance of
vaccines for birds and for diagnostic, virus load on samples that are positive.
