Commercial RT-PCR Assay Research Articles

Evaluation of 16 molecular assays for the detection of orthopox and mpox viruses

The current global mpox virus (MPXV) outbreak has been declared a Public Health Emergency of International Concern by WHO, with more than 80,000 cases confirmed across multiple continents. Diagnosis is confirmed by PCR of viral DNA from vesicle and other swabs. The aim of this study was to assess commercial RT PCR assays for Orthopoxvirus (OPX) and MPXV for analytical sensitivity, and percent agreements and compare them to primer/probe sets employed at the Victorian Infectious Diseases Reference Laboratory (VIDRL), Centers for Disease Control andPrevention (CDC) and US Army Medical Research Institute of Infectious Diseases (USAMRIID). Limits of detection (LOD), intra-run variability, cross-reactivity and performance on forty clinical samples was assessed on eleven commercial assays and five primer/probe combinations used at VIDRL, CDC and USAMRIID. All assays were able to detect OPX and MPXV (LOD 57 to 14,495 copies/mL) with intra-run coefficients of variation between Cycle thresholds of 0.58 and 3.44, and there was no unexpected cross-reactivity. All assays demonstrated 100% negative percent agreement with clinical samples and all but one yielded 100% positive percent agreement. Variations in LOD between assays may be dependent on the platform used and sample type. Despite the overall comparable performance of the assays assessed, it is important that routine laboratories perform in-house validations before implementing RT PCR for OPX and/or MPXV as reliable and accurate laboratory diagnosis of MPXV and isolation is crucial to containing the spread of this current outbreak and informing public health interventions and response.

Investigating the sequence variation in the influenza A matrix genes during the 2017-2018 and 2018-2019 seasons in samples from a local population in London

Recent publications have highlighted the emergence of mutations in the M1 gene of both influenza A H1N1pdm09 and H3N2 subtypes affecting the performance of commercial RT-PCR assays. Respiratory samples from the 2018/2019 season positive by our in-house RT-PCR for influenza A were analysed for the prevalence and impact of any M1 gene mutations. Sequence information was used to re-design primers for our routine assay and their performance assessed. Forty-five samples, consisting of 11 H1N1pdm09 and 34 H3N2 subtypes, together with the NIBSC H1N1 control were sequenced. All samples displayed the core mutations for H1N1 M1(C154T; G174A and G238A) and for H3N2 M1(C153T; C163T and G189T); three of the H1N1pdm09 viruses also showed a small number of point mutations. None of the mutations appeared to affect either the sensitivity or efficiency of the RT-PCR when compared to the re-designed primers. Although the mutations we found agreed with those in the publications cited we did not encounter any problems with our routine diagnostic assay and no improvements were found when the primers were modified to suit those mutations. However, it is likely that the influenza A virus M1 gene will accumulate further mutations that could impact RT-PCR assays and, therefore, it would be prudent to implement routine sequencing of samples during the influenza seasons to ensure no loss in assay performance.

Usefulness of the GenMark ePlex RPP assay for the detection of respiratory viruses compared to the FTD21 multiplex RT-PCR

Cartridge-based multiplex panels covering numerous pathogens offer an advantage of minimal hands-on-time and short time to result to commercial RT-PCR assays. In this study, we evaluated the performance of the ePlex respiratory pathogen panel (RPP) compared to the Fast Track Diagnostics Respiratory pathogens 21 multiplex RT-PCR assay (FTD21) using 400 clinical respiratory samples. Discrepant results were further analysed by a reference nucleic acid amplification testing (NAT) and a composite reference approach was used for final interpretation. Discordant results were observed in 56 targets corresponding to 54 samples. Sensitivities and specificities were 85.5% and 99.9% for the ePlex RPP and 95.8% and 99.7% for the FTD21 system, respectively.Altogether, the ePlex RPP is a valuable tool for the rapid detection of a number of different respiratory viruses with the exception of the coronavirus family (low sensitivity ranging from 50-80%) and samples with a low pathogen load (Ct values >33).

Development of a TaqMan loop-mediated isothermal amplification assay for the rapid detection of pigeon paramyxovirus type 1.

Pigeon paramyxovirus-1 (PPMV-1) is a strain of Newcastle disease virus (NDV) that has adapted to infect pigeons and poses a constant threat to the commercial poultry industry. Early detection via rapid and sensitive methods, along with timely preventative and mitigating actions, is important for reducing the spread of PPMV-1. Here, we report the development of a TaqMan loop-mediated isothermal amplification assay (TaqMan-LAMP) for rapid and specific detection of PPMV-1 based on the F gene. This system makes use of six novel primers and a TaqMan probe that targets nine distinct regions of the F gene that are highly conserved among PPMV-1 isolates. The results showed that the limit of detection was 10 copies μL-1 for PPMV-1 cDNA and 0.1 ng for PPMV-1 RNA. The reaction was completed within 25 min and was thus faster than conventional RT-PCR. Moreover, no cross-reactions with similar viruses or with peste des petits ruminants virus (PPRV) or NDV LaSota vaccine strains were observed under the same conditions. To evaluate the applicability of the assay, the TaqMan-LAMP assay and a commercial RT-PCR assay were compared using 108 clinical samples, and the concordance rate between two methods was found to be 96.3%. The newly developed PPMV-1 TaqMan-LAMP assay can therefore be used for simple, efficient, rapid, specific, and sensitive diagnosis of PPMV-1 infections.

Comparison of in-house and commercial real time-PCR based carbapenemase gene detection methods in Enterobacteriaceae and non-fermenting gram-negative bacterial isolates

BackgroundCarbapenemase-producing gram-negative bacteria are increasing globally and have been associated with outbreaks in hospital settings. Thus, the accurate detection of these bacteria in infections is mandatory for administering the adequate therapy and infection control measures. This study aimed to establish and evaluate a multiplex real-time PCR assay for the simultaneous detection of carbapenemase gene variants in gram-negative rods and to compare the performance with a commercial RT-PCR assay (Check-Direct CPE).Methods116 carbapenem-resistant Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii isolates were genotyped for carbapenemase genes by PCR and sequencing. The defined isolates were used for the validation of the in-house RT-PCR by use of designed primer pairs and probes.ResultsAmong the carbapenem-resistant isolates the genes blaKPC, blaVIM, blaNDM or blaOXA were detected. Both RT-PCR assays detected all blaKPC, blaVIM and blaNDM in the isolates. The in-house RT-PCR detected 53 of 67 (79.0%) whereas the commercial assay detected only 29 (43.3%) of the OXA genes. The in-house sufficiently distinguished the most prevalent OXA types (23-like and 48-like) in the melting curve analysis and direct detection of the genes from positive blood culture vials.ConclusionThe Check-Direct CPE and the in-house RT-PCR assay detected the carbapenem resistance from solid culture isolates. Moreover, the in-house assay enabled the identification of carbapenemase genes directly from positive blood-culture vials. However, we observed insufficient detection of various OXA genes in both assays. Nevertheless, the in-house RT-PCR detected the majority of the OXA type genes in Enterobacteriaceae and A. baumannii.

Detection of methylated septin 9 in tissue and plasma of colorectal patients with neoplasia and the relationship to the amount of circulating cell-free DNA.

BackgroundDetermination of methylated Septin 9 (mSEPT9) in plasma has been shown to be a sensitive and specific biomarker for colorectal cancer (CRC). However, the relationship between methylated DNA in plasma and colon tissue of the same subjects has not been reported.MethodsPlasma and matching biopsy samples were collected from 24 patients with no evidence of disease (NED), 26 patients with adenoma and 34 patients with CRC. Following bisulfite conversion of DNA a commercial RT-PCR assay was used to determine the total amount of DNA in each sample and the fraction of mSEPT9 DNA. The Septin-9 protein was assessed using immunohistochemistry.ResultsThe percent of methylated reference (PMR) values for SEPT9 above a PMR threshold of 1% were detected in 4.2% (1/24) of NED, 100% (26/26) of adenoma and 97.1% (33/34) of CRC tissues. PMR differences between NED vs. adenoma and NED vs. CRC comparisons were significant (p<0.001). In matching plasma samples using a PMR cut-off level of 0.01%, SEPT9 methylation was 8.3% (2/24) of NED, 30.8% (8/26) of adenoma and 88.2% (30/34) of CRC. Significant PMR differences were observed between NED vs. CRC (p<0.01) and adenoma vs. CRC (p<0.01). Significant differences (p<0.01) were found in the amount of cfDNA (circulating cell-free DNA) between NED and CRC, and a modest correlation was observed between mSEPT9 concentration and cfDNA of cancer (R2 = 0.48). The level of Septin-9 protein in tissues was inversely correlated to mSEPT9 levels with abundant expression in normals, and diminished expression in adenomas and tumors.ConclusionsMethylated SEPT9 was detected in all tissue samples. In plasma samples, elevated mSEPT9 values were detected in CRC, but not in adenomas. Tissue levels of mSEPT9 alone are not sufficient to predict mSEPT9 levels in plasma. Additional parameters including the amount of cfDNA in plasma appear to also play a role.

Evaluation of novel H1N1-specific primer-probe sets using commercial RT-PCR mixtures and a premixed reaction stored in a lyophilized format

The recent emergence of a novel H1N1 influenza A virus in humans caused the first influenza pandemic of this century. Many clinical diagnostic laboratories are overwhelmed by the testing demands related to the infection. Three novel H1N1-specific primer-probe sets reported during the early phase of the pandemic were tested in three commercial real-time RT-PCR mixtures. The amplification efficiencies and detection limits of these assays were determined. A ready-to-use premixed RT-PCR stored in a lyophilized format was developed. The detection limits of the studied assays were highly variable, ranging from 1.68E−01 to 1.68E−05 TCID 50 per reaction. The detection limit of the lyophilized reaction mixture was found to be 1.68E−05 TCID 50 per reaction, but the amplification efficiency of the assay was lower than those deduced from the other assays. All respiratory samples from infected patients and all control nasopharyngeal aspirates were positive and negative, respectively, in the newly developed assay. The results highlighted that, to enhance the sensitivity of an assay, it is essential to evaluate a primer-probe set with different commercial RT-PCR assays. This study also demonstrated the feasibility of using lyophilized reaction mixtures for the molecular diagnosis of novel H1N1.

The number of HIV DNA-infected mononuclear cells is reduced under HAART plus recombinant IL-2

It is common opinion that, in addition to potent antiretroviral regimens which effectively reduce plasma viremia, new strategies should be developed to ensure the reduction of cell-associated HIV DNA load together with HIV RNA plasma levels. The present study explored whether the number of provirus-infected cells can be reduced by combined antiviral and immunomodulatory regimens. Thus, 14 naive patients (with CD4 >400/μl and plasma HIV RNA copies>5000/ml) were randomly assigned to receive highly active antiretroviral therapy (HAART) alone or HAART plus rIL-2. Plasma viremia (measured by a commercial RT-PCR assay) and the number of provirus-infected cells (measured by an endpoint cell dilution PCR assay) were monitored at the enrollment and after 12 weeks of treatment. The results indicate that while HAART and HAART plus rIL-2 are both able to significantly reduce plasma viremia after 12 weeks of treatment, a significant reduction of the number of provirus-infected cells can be achieved only by treatment with HAART plus rIL-2.

Seroreactivity clarification and viral load quantitation in HIV-1 and HIV-2 infections in Ghana.

In Ghana, West Africa, the prevalence of dual HIV-1 and HIV-2 infections remains to be clarified, and HIV viral load measurement is yet to be established. Conventional assays for HIV-1 RNA measurements have been limited specifically to HIV-1 subtype B, preventing their utilization for Ghana where HIV-1 subtypes A, D and G are prevalent. Therefore, we set out to distinguish the types of HIV infection existing in Ghana so as to determine the extent of actual dual infections, and to measure plasma HIV-1 RNA. Blood samples were collected from 563 sick and healthy Ghanaians who visited hospitals in 1996 and 1997. After T cells were counted, HIV antibody was screened and confirmed by six different commercial assays and one in-house test. Nested PCR was then used to verify HIV-1 and HIV-2 presence by type-specific primers. Plasma HIV-1 RNA was measured by an improved commercial RT-PCR assay, sensitive to all HIV-1 group M subtypes. HIV-1 alone (89%) clearly dominated over HIV-2 alone (2%), and HIV-1 and HIV-2 dual infections were found in 9%. Valid viral load measurements were obtained on test plasma representing the main HIV-1 subtype (A) prevailing in Ghana. A high amount of HIV-1 RNA (5.9 mean log10 RNA copies/ml) was observed in the typical stages of HIV infection represented by groups of CD44 cell counts. We have clarified the seroprevalence of HIV-1 and HIV-2 amongst HIV seropositives, and the high viral load of HIV-1 reflects its influence on AIDS in Ghana.