Validation of Synovial Fluid Clinical Samples for Molecular Detection of Pathogens Causing Prosthetic Joint Infection Using GAPDH Housekeeping Gene as Internal Control

Abstract

Identification of the pathogens causing infection is important in terms of patient's health management and infection control. Synovial fluids could be used as clinical samples to detect causative pathogens of prosthetic joint infections (PJIs) using molecular diagnostic assays, therefore, normalization and validation of clinical samples are necessary. Microbial culture is considered the gold standard for all infections, including PJIs. Recently, molecular diagnostic methods have been developed to overcome the limitation of microbial culture. Therefore, guideline for validating clinical samples to provide reliable results of molecular diagnostic assays for infectious diseases is required in clinical field. The present study aimed to develop an accurate validating method of synovial fluid clinical samples using GAPDH gene as an internal control to perform the quantitative PCR TaqMan probe assay to detect pathogens causing PJIs.

Diagnostic Performance of a Molecular Assay in Synovial Fluid Targeting Dominant Prosthetic Joint Infection Pathogens

“…To monitor the efficiency of nucleic acid extraction and the potential presence of PCR inhibitors during the analysis, primer and hydrolysis probe sets were designed to detect GAPDH gene as an internal control. All 93 SF samples were tested using GAPDH primers and hydrolysis probes described elsewhere [ 23 ] and validated using GAPDH qPCR. In total, 45.16% (42/93) were GAPDH -negative and were therefore omitted from analysis, and the 51 (54.84%) GAPDH -positive samples were used for analysis.…”

“…In this study, we developed a qPCR diagnostic method to detect bacteria and fungi in SF using 16S rRNA and 18S rRNA genes, respectively, and evaluated its effectiveness. Prior to evaluating the qPCR diagnostic method, we had already established an accurate validation method for clinical SF samples using GAPDH as an internal control [ 23 ], which was then applied to all SF samples. In another study using GAPDH primers as an internal control, qPCR was performed, and nonamplified samples were excluded from case analysis [ 41 ].…”

“…To validate clinical SF samples, the glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) housekeeping gene was amplified using a primer–hydrolysis probe set [ 23 ]. GAPDH -negative samples with a Ct value ≥ 30 were omitted from the analysis ( Table 1 ).…”