Development and validation of a reverse transcription real-time PCR assay for specific detection of PRRSGard vaccine-like virus.

Increasing use of modified live virus (MLV) vaccines presents challenges to interpret positive results of porcine reproductive and respiratory syndrome virus (PRRSV) screening PCR that can detect both wild-type and vaccine strains. Instead, vaccine-specific PCR provides a convenient tool to detect vaccine-like virus from a sample. Here we report the development and validation of a real-time RT-PCR specific for PRRSGard® , a newly available commercial PRRSV-2 MLV vaccine. Analytical specificity, sensitivity and diagnostic performance of PRRSGard PCR were evaluated and compared to a commercial PRRSV screening PCR (reference PCR). PRRSGard and reference PCRs did not cross-react with any of the 27 non-PRRSV swine pathogens. PRRSGard PCR did not cross-react with other PRRSV-2 vaccine viruses and 31 laboratory and field PRRSV-2 isolates representing various genetic lineages of PRRSV-2. PRRSGard and reference PCRs consistently detected up to 10-6 and 10-5 dilutions of PRRSGard vaccine virus, respectively. Based on testing serial dilutions of in vitro transcribed RNA, the 95% limit of detection of PRRSGard PCR was 16 genomic copies/reaction with CT cut-off value of 36 and 7 genomic copies/reaction with CT cut-off value of 37. Diagnostic performance of PRRSGard PCR was evaluated using 846 clinical samples (684 serum and 162 oral fluid samples). Compared to the reference screening PCR, diagnostic sensitivity, specificity and agreement of PRRSGard PCR were 95.34%, 98.85% and 97.52% with cut-off CT value of 36 and 98.14%, 96.56% and 97.16% with cut-off CT value of 37. In addition, PRRSGard PCR was able to detect PRRSGard vaccine virus in a sample even with the co-presence of another PRRSV strain. In summary, in contrast to a reference screening PCR that detects both vaccine and field PRRSV strains, PRRSGard PCR provides a convenient tool to specifically detect PRRSGard vaccine-like virus and to inform PRRSV vaccination protocols.

Methicillin-resistant Staphylococcus aureus Screening PCR adapted to locally emerging variants-Evaluation of novel SCCmec primers.

Infections with multi-resistant bacteria, such as Methicillin-resistant Staphylococcus aureus (MRSA), represent a world-wide health-care problem. The original MRSA Screening TaqMan PCR was based on the detection of the SCCmec-orfX-junction as described by the group of Huletsky in 2004. In the recent years, this assay increasingly failed to detect new MRSA variants in swab specimens. In this work, we analyzed the usefulness of 17 additional SCCmec primers to increase PCR sensitivity by testing 290 collected samples with negative PCR results and positive MRSA culture in a retrospective analysis, and 380 samples of the daily routine diagnostics. Sequencing of the PCR products revealed that locally new MRSA variants became detectable by nine of these forward primers. Four primers were solely responsible for the detection of 85.4% (117/123) of the PCR products: F13 (n=76), F11 (n=6), F14 (n=15) and F25 (n=8). These four primers were integrated in the Screening PCR and the novel primer collection was validated by testing 71 MRSA isolates, which covered SCCmec types I to VI, 50 MSSA isolates and 100 swab specimens. The sensitivity of MRSA Screening PCR increased from 93% to 98.6% without affecting the detection of the common MRSA strains. Phylogenetic analysis of the PCR products suggests that the adapted MRSA Screening PCR is able to detect SCCmec types I-X, including CA- and LA-MRSA variants by the SCCmec primers F11 and F25.

Towards a Better Molecular Diagnosis of FMR1-Related Disorders-A Multiyear Experience from a Reference Lab.

The article summarizes over 20 years of experience of a reference lab in fragile X mental retardation 1 gene (FMR1) molecular analysis in the molecular diagnosis of fragile X spectrum disorders. This includes fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency (FXPOI) and fragile X-associated tremor/ataxia syndrome (FXTAS), which are three different clinical conditions with the same molecular background. They are all associated with an expansion of CGG repeats in the 5'UTR of FMR1 gene. Until 2016, the FMR1 gene was tested in 9185 individuals with the pre-screening PCR, supplemented with Southern blot analysis and/or Triplet Repeat Primed PCR based method. This approach allowed us to confirm the diagnosis of FXS, FXPOI FXTAS in 636/9131 (6.96%), 4/43 (9.3%) and 3/11 (27.3%) of the studied cases, respectively. Moreover, the FXS carrier status was established in 389 individuals. The technical aspect of the molecular analysis is very important in diagnosis of FXS-related disorders. The new methods were subsequently implemented in our laboratory. This allowed the significance of the Southern blot technique to be decreased until its complete withdrawal. Our experience points out the necessity of implementation of the GeneScan based methods to simplify the testing procedure as well as to obtain more information for the patient, especially if TP-PCR based methods are used.

Screening PCR Versus Sanger Sequencing: Detection of CALR Mutations in Patients With Thrombocytosis.

BACKGROUND: Mutations in calreticulin (CALR) have been reported to be key markers in the molecular diagnosis of myeloid proliferative neoplasms. In most previous reports, CALR mutations were analyzed by using Sanger sequencing. Here, we report a new, rapid, and convenient system for screening CALR mutations without sequencing. METHODS: Eighty-three bone marrow samples were obtained from 81 patients with thrombocytosis. PCR primers were designed to detect wild-type CALR (product: 357 bp) and CALR with type 1 (product: 302 bp) and type 2 mutations (product: 272 bp) in one reaction. The results were confirmed by Sanger sequencing and compared with results from fragment analysis. RESULTS: The minimum detection limit of the screening PCR was 10 ng for type 1, 1 ng for type 2, and 0.1 ng for cases with both mutations. CALR type 1 and type 2 mutants were detected with screening PCR with a maximal analytical sensitivity of 3.2% and <0.8%, respectively. The screening PCR detected 94.1% (16/17) of mutation cases and showed concordant results with sequencing in the cases of type 1 and type 2 mutations. Sanger sequencing identified one novel mutation (c.1123_1132delinsTGC). Compared with sequencing, the screening PCR showed 94.1% sensitivity, 100.0% specificity, 100.0% positive predictive value, and 98.5% negative predictive value. Compared with fragment analysis, the screening PCR presented 88.9% sensitivity and 100.0% specificity. CONCLUSIONS: This screening PCR is a rapid, sensitive, and cost-effective method for the detection of major CALR mutations.

Screening PCR Versus Sanger Sequencing: Detection of CALR Mutations in Patients With Thrombocytosis

BACKGROUND: Mutations in calreticulin (CALR) have been reported to be key markers in the molecular diagnosis of myeloid proliferative neoplasms. In most previous reports, CALR mutations were analyzed by using Sanger sequencing. Here, we report a new, rapid, and convenient system for screening CALR mutations without sequencing. METHODS: Eighty-three bone marrow samples were obtained from 81 patients with thrombocytosis. PCR primers were designed to detect wild-type CALR (product: 357 bp) and CALR with type 1 (product: 302 bp) and type 2 mutations (product: 272 bp) in one reaction. The results were confirmed by Sanger sequencing and compared with results from fragment analysis. RESULTS: The minimum detection limit of the screening PCR was 10 ng for type 1, 1 ng for type 2, and 0.1 ng for cases with both mutations. CALR type 1 and type 2 mutants were detected with screening PCR with a maximal analytical sensitivity of 3.2% and <0.8%, respectively. The screening PCR detected 94.1% (16/17) of mutation cases and showed concordant results with sequencing in the cases of type 1 and type 2 mutations. Sanger sequencing identified one novel mutation (c.1123_1132delinsTGC). Compared with sequencing, the screening PCR showed 94.1% sensitivity, 100.0% specificity, 100.0% positive predictive value, and 98.5% negative predictive value. Compared with fragment analysis, the screening PCR presented 88.9% sensitivity and 100.0% specificity. CONCLUSIONS: This screening PCR is a rapid, sensitive, and cost-effective method for the detection of major CALR mutations.