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PubMed2026

Saliva versus lesion swabs for PCR diagnosis of acute-phase clade Ib mpox in Uganda: a prospective matched hospital cohort study.

BACKGROUND: As clade Ib mpox expands through HIV-affected populations in east and central Africa, diagnostic specimen selection should balance accuracy, accessibility, and operational feasibility in outbreak settings. Here, we aimed to compare the diagnostic performance of matched plasma, saliva, genital, anal, and skin specimens during the acute rash phase of clade Ib mpox to identify clinically practical and high-yield sampling approaches for outbreak response and clinical care. METHODS: We conducted a prospective cohort study of 155 adults (median age 30 years, IQR 25-36) hospitalised at Uganda's national mpox referral hospital. The specimens were collected between March 3 and April 10, 2025, during the clade Ib outbreak. All participants were admitted with suspected mpox and were subsequently confirmed by MPXV PCR. We collected 836 clinical specimens (acid citrate dextrose plasma, saliva, genital swabs, anal swabs, and skin swabs) during the acute phase (visit 1; 14 days [SD 2] after systemic symptom onset) and at approximately 3 months (visit 2). A matched acute-phase subset (n=80) provided concurrent plasma, saliva, genital, and skin specimens for within-participant comparisons. MPXV DNA was quantified by F3L real-time quantitative PCR, and cycle threshold (Ct) values were compared using paired Wilcoxon signed-rank tests. Whole-genome sequencing of selected acute specimens confirmed clade assignment. FINDINGS: In the matched subset at visit 1, PCR positivity was high in saliva (78 [98%] of 80), skin swabs (78 [98%] of 80), and genital swabs (77 [96%] of 80). Results for the saliva closely mirrored genital and skin swab results, supporting saliva as a high-yield alternative when lesion sampling is painful, operationally difficult, or unacceptable. Plasma had substantially lower sensitivity (34 [43%] of 80) and showed poor agreement with mucocutaneous compartments. At 3 months, persistent MPXV DNA was rare (ten [9%] of 109) and clustered among people with HIV, including the only two participants with persistent plasma positivity. All sequenced genomes clustered within clade Ib. INTERPRETATION: During the established rash phase (14 days [SD 2] after onset), saliva provides diagnostic yield similar to that provided by lesion swabs for clade Ib mpox in this hospitalised cohort. These findings are restricted to this sampling window; further studies are needed to define performance in prodromal, pre-rash, and asymptomatic infection. FUNDING: CEPI through its Centralised Laboratory Network.

Adult↗
PubMed2006

Evaluation of uncertainty in quantitative real-time PCR.

Quantitative real-time PCR is one of the newer methods for measurement of the amount of nucleic material in biological systems. However, reliable measurement requires an appropriate estimation of uncertainty and this paper has developed the uncertainty budget associated with this procedure using as an example, data from a quantitative real-time PCR method for the enumeration of Campylobacter jejuni. This uncertainty is relatively large and for instance, a measured result of 151 units of DNA would have a 95% confidence interval of +/-84 units of DNA with the main sources of uncertainty being the measurement of the threshold cycle (Ct) value, the predicted DNA content of the unknown sample from the calibration line and the molar absorbance value for DNA.

Campylobacter jejuni↗
PubMed2005

Development of a sensitive and quantitative diagnostic assay for fish nervous necrosis virus based on two-target real-time PCR.

The aim of the present work was to develop two new independent SYBR Green I-based real-time PCR assays for both detection and quantification of betanodavirus, an RNA virus that infects several species of marine teleost fish causing massive mortalities in larvae and juveniles. The assays utilized two pairs of primers targeting highly conserved regions of both the RNA molecules forming the betanodavirus genome: RNA1 encoding the RNA-dependent RNA polymerase (RdRP) and RNA2 encoding the coat protein (CP). The specificity of amplifications was monitored by the melting analysis and agarose gel electrophoresis of the amplified products. The applicability of these assays was confirmed with 21 betanodavirus strains, covering all the four main clades. In addition, a BLAST (NCBI) search with the primer sequences showed no genomic cross-reactivity with other viruses. The new assays were able to quantify concentrations of betanodavirus genes ranging from 10(1) to 10(8) copies per reaction. The intra-assay coefficients of variation (CV) of threshold cycle (Ct) values of the assays were 1.5% and 1.4% for CP and RdRP RNAs, respectively. The inter-assay CVs of Ct values were 2.3% and 2.4% for CP and RdRP RNAs, respectively. Moreover, regression analysis showed a significant correlation (R2>0.97) between genome number, as determined by real-time PCR assays and the corresponding virus titer expressed as TCID50/ml of two different betanodavirus strains propagated in cell culture. The two assays were compared with a previously established one-step RT-PCR assay and with the classical virus isolation test and found to be more sensitive. In conclusion, the developed real-time RT-PCR assays are a reliable, specific and sensitive tool for the quantitative diagnosis of betanodavirus.

Animals↗
PubMed2004

Establishment of a real-time RT-PCR for the determination of absolute amounts of IGF-I and IGF-II gene expression in liver and extrahepatic sites of the tilapia.

We developed a one-tube two-temperature real-time RT-PCR that allows to absolutely quantify the gene expression of hormones using the standard curve method. As our research focuses on the expression of the insulin-like growth factors (IGFs) in bony fish, we established the technique for IGF-I and IGF-II using the tilapia (Oreochromis niloticus) as model species. As approach, we used primer extension adding a T7 phage polymerase promoter (21 nt) to the 5' end of the antisense primers. This procedure avoids the disadvantages arising from plasmids. Total RNA extracted from liver was subjected to conventional RT-PCR to create templates for in vitro transcription of IGF-I and IGF-II cRNA. Correct template sizes including the T7 promoter were verified (IGF-I: 91 nt; IGF-II: 94 nt). The PCR products were used to create IGF-I and IGF-II cRNAs which were quantified in dot blot by comparison with defined amounts of standardised kanamycin mRNA. Standardised threshold cycle (Ct) values for IGF-I and IGF-II mRNA were achieved by real-time RT-PCR and used to create standard curves. To allow sample normalisation the standard curve was also established for beta-actin as internal calibrator (template: 86 nt), and validation experiments were performed demonstrating similar amplification efficiencies for target and reference genes. Based on the standard curves, the absolute amounts of IGF-I and IGF-II mRNA were determined for liver (IGF-I: 8.90+/-1.90 pg/microg total RNA, IGF-II: 3.59+/-0.98 pg/microg total RNA) and extrahepatic sites, such as heart, kidney, intestine, spleen, gills, gonad, and brain considering the different lengths of cRNAs and mRNAs by correction factors. The reliability of the method was confirmed in additional experiments. The amplification of descending dilutions of cRNA and total liver RNA resulted in parallel slopes of the amplification curves. Furthermore, amplification plots of the standard cRNA and the IGF-I and IGF-II mRNAs showed signals starting at the expected Ct values. Thus, the one-tube RT-PCR described here is highly sensitive (detection level approximately 2 pg/microg total RNA) and allows precise absolute quantification. The method is rapid as there are neither separate reverse transcriptions nor post-amplification steps, and can be executed with low risk of contamination. Therefore, it will be helpful when investigating gene expression in any species and tissue whenever absolute levels are of concern.

Animals↗
PubMed2002

Real-time RT-PCR for quantitation of hepatitis C virus RNA.

A newly developed real-time RT-polymerase chain reaction assay for quantitation of hepatitis C virus (HCV) RNA in human plasma and serum was applied. A pair of primers and a probe (molecular beacon) were designed that are specific for the recognition of a highly conservative 5'-non-coding region (5'-NCR) in HCV genome. HCV real-time RT-PCR assay had a sensitivity of 1000 RNA copies per reaction, with a dynamic range of detection between 10(3) and 10(7) RNA copies. The coefficient variation of threshold cycle (Ct) values in intra- and inter-runs were less than 1.37 and 4.66%, respectively. The real-time RT-PCR assay on the HCV sero-positive samples yielded reproducible data, with less than 2.09% of the inter-assay variation. In order to determine its potential for clinical diagnosis, real-time RT-PCR was used to examine the HCV RNA levels in plasma from sero-positive and negative subjects, showing that the assay is highly sensitive and has specificity of 100%. It was demonstrated that the real-time RT-PCR was able to amplify HCV RNA in reference sera with seven genotypes (1A, 1B, 2B, 3A, 4, 5A and 6A) that include six major HCV genotypes circulated in the world. Since HCV is a major pathogen of post-transfusion and community-transmitted non-A, non-B hepatitis, this assay has a broad application for basic and clinical investigations.

5' Untranslated Regions↗
PubMed2003

A quantitative method to detect HBV cccDNA by chimeric primer and real-time polymerase chain reaction.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), is a continuous double chain ring, while other DNA forms contain a gap on each chain at the position of direct repeat 1 sequence (DR1) and direct repeat 2 sequence (DR2), respectively. At present it is still difficult to detect with high sensitivity and specificity and quantify the HBV cccDNA pool in the nucleus of the hepatocyte. A chimeric primer was designated in which segment A near 3' end is complementary to HBV plus strand just before the DR2 region gap, and segment B near 5' end is consensus to part of the human immunodefficient virus genomic sequence, without homogenetic relationship to HBV genome. Promoted by taq DNA polymerase, a single nucleotide strand is elongated from chimeric primer generated by HBV cccDNA. In contrast, other HBV DNA forms do not produce a single nucleotide strand due to the cessation of elongation reaction at the DR2 gap. The newly formed single nucleotide strand is subsequently amplified by a new polymerase chain reaction system (PCR), in which a primer, identical to chimeric primer segments B, is used to ensure specific amplification, avoiding other HBV DNA format inference. In addition, a taqman probe was used in the PCR system to report the detection signal, and for constructing a standard curve between cycle threshold (Ct) value and the template quantity. This technique proved to be effective for rapid and sensitive detection and quantitation of HBV cccDNA with high specificity and efficacy.

DNA Primers↗
PubMed2003

The presence of a significant association between elevated PRV-1 mRNA expression and low plasma erythropoietin concentration in essential thrombocythaemia.

Approximately 45% of newly diagnosed patients with essential thrombocythaemia (ET) demonstrate subnormal plasma erythropoietin (EPO) concentrations, which constitutes a risk factor for occlusive vascular events. In 58 ET patients, a possible association between polycythaemia rubra vera-1 (PRV-1) overexpression and subnormal plasma EPO was investigated, which was always measured prior to the institution of platelet lowering agents. At the time when PRV-1 expression was measured, 28 of 58 (48%) ET patients had received platelet lowering treatment. PRV-1 expression was measured by quantitative real-time reverse transcription-polymerase chain reaction assay of mRNA extracted from purified peripheral blood buffy coat. The cycle threshold (CT) value of PRV-1 was determined and was divided with the CT value for the housekeeping GAPDH gene transcript. A quotient <0.93 was defined as PRV-1 positive. Of the ET patients 12 of 58 (21%) were PRV-1 positive and 19 of 58 (33%) demonstrated subnormal plasma EPO. In the 58 ET patients there was a significant association between low plasma EPO and PRV-1 positive results (P = 0.001). The 30 ET patients who had not received any platelet lowering treatment showed a significant (P = 0.005) relation between PRV-1 positivity and subnormal plasma EPO. No such relationship was present in the 28 ET patients who had received prior treatment with the above drugs (P = 0.147).

Adult↗
PubMed2000

Application of germline IGH probes in real-time quantitative PCR for the detection of minimal residual disease in acute lymphoblastic leukemia.

Large-scale clinical studies on detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL) have shown that quantification of MRD levels is needed for reliable MRD-based risk group classification. Recently, we have shown that 'real-time' quantitative PCR (RQ-PCR) can be applied for this purpose using patient-specific immunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements as PCR targets with TaqMan probes at the position of the junctional region and two germline primers. Now, we tested an alternative approach on 35 immunoglobulin heavy chain (IGH) gene rearrangements, by designing three germline JH TaqMan probes to be used in combination with one of six corresponding germline JH primers and one allele specific oligonucleotide (ASO) primer complementary to the junctional region. In nine cases in which both approaches were compared, at least similar (n = 4) or slightly higher (n= 5) maximal sensitivities were obtained using an ASO primer. The ASO primer approach reached maximal sensitivities of at least 10(-4) in 33 out of 35 IGH rearrangements. The reproducible range for accurate quantification spanned four to five orders of magnitude in 31 out of 35 cases. In 13 out of 35 rearrangements the stringency of PCR conditions had to be increased to remove or diminish background signals; this only concerned the frequently occurring JH4, JH5 and JH6 gene rearrangements. After optimization of the conditions (mainly by increasing the annealing temperature), only occasional aspecific amplification signals were observed at high threshold cycle (CT) values above 42 cycles and at least six cycles above the CT value of the detection limit. Hence, these rare aspecific signals could be easily discriminated from specific signals. We conclude that the here presented set of three germline JH Taq-Man probes and six corresponding germline JH primers can be used to develop patient-specific RQ-PCR assays, which allow accurate and sensitive MRD analysis in almost all IGH gene rearrangements. These results will facilitate standardized RQ-PCR analysis for MRD detection in large clinical studies.

DNA Probes↗
PubMed2001

Increased endothelin-1 and decreased adrenomedullin gene expression in the stenotic tissue of congenital pelvi-ureteric junction obstruction in children.

OBJECTIVES: To test the hypothesis that the gene expression of endothelin-1 and adrenomedullin may be altered in stenotic tissues of patients with congenital hydronephrosis caused by pelvi-ureteric junction (PUJ) obstruction. MATERIALS AND METHODS: Using real-time reverse transcription-polymerase chain reaction, mRNA of smooth muscle-constricting endothelin-1 and of smooth muscle-relaxing adrenomedullin was quantified in tissue specimens of 20 patients with PUJ obstruction (mean age 5.1 years, SD 7.0) and of 21 controls with normal PUJs (mean age 23.5 years, SD 24.2). RESULTS: The amount of endothelin-1 mRNA in stenotic specimens was higher than in the controls, indicated by significantly lower threshold cycles (Ct values) in real-time PCR for the target gene in the obstructive tissue, with mean (SD) values of 24.9 (1.6) and 26.0 (2.1) (P < 0.05), respectively. The endothelin-1/CD31 ratio was significantly higher in the patients (P < 0.05) than in controls. In addition, adrenomedullin gene expression in the obstructed junctions was significantly lower than in normal junctions, with higher Ct values for the patient group of 26.7 (1.6) vs 25.2 (1.8) (P < 0.05) and lower adrenomedullin mRNA when standardized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (P < 0.05), CD31 (P < 0.01) and smooth muscle alpha-actin mRNA (P < 0.01). The two groups showed no significant differences for GAPDH and CD31 mRNA content, whereas there was about twice as much alpha-actin mRNA in stenotic tissues than in unaffected PUJs, shown by the lower Ct values for the patient group of 16.9 (2.0) vs 17.9 (2.6) (P < 0.05). Furthermore, endothelin-1, adrenomedullin and alpha-actin mRNA amounts were independent of age. CONCLUSION: Taken together these results provide evidence that the production of autocrine/paracrine acting endothelin-1 and adrenomedullin is altered in tissues of patients with genuine PUJ obstruction, and may be involved in the pathogenesis of congenital hydronephrosis.

Adolescent↗
PubMed2006

Comparison of real-time polymerase chain reaction and end-point polymerase chain reaction for the analysis of gene expression in preimplantation embryos.

The aim of the present study was to compare real-time polymerase chain reaction (PCR) and end-point PCR with respect to their suitability for the analysis of gene expression in samples in which the number of cells is limited; for example, in studies of preimplantation embryonic development and to determine the variability of the real-time reverse transcription-PCR assay. The sensitivity, dynamic range and precision of both PCR systems were compared using a single mouse liver cDNA standard. The real-time system was 100-fold more sensitive than the end-point system and had a dynamic range of more than four orders of magnitude. The linear range for end-point PCR extended for two orders of magnitude using a fixed end-point of 31 cycles. The percentage standard error of the mean based on 30 replicates was 0.14% of the threshold cycle (Ct) value for the real-time system and 6.8% for the end-point fluorescence intensity. The coefficients of variation (CV) for reverse transcription combined with real-time analysis and the complete gene expression protocol consisting of mRNA isolation, reverse transcription and real-time PCR analysis were 0.6% and 1.4% of the Ct values, respectively. The present paper details, for the first time, measurement of the biological variation of individual mammalian oocytes. The CV was 1.8% of the Ct value for expression analysis of six bovine oocytes. The results are discussed in relation to the analysis of gene expression in preimplantation embryo development.

Animals↗
PubMed2004

Reduction of connexin 43 expression in aged human dental pulp.

AIM: To investigate the expression of connexin 43 (CX43) mRNA in young and old human dental pulp tissues to determine the characteristics of CX43 expression. METHODOLOGY: Samples were obtained from human dental pulp of healthy young (17-23 years) and aged (>50 years) subjects. CX43 expression was determined by RT-PCR and by quantitative real-time RT-PCR (QRT-PCR). The threshold cycle (Ct) value, which reflects the amount of PCR, was calculated and the difference between value in the young pulp and that in the aged pulp was statistically analysed. RESULTS: RT-PCR analysis of human dental pulp tissue detected CX43 mRNA in all the samples. CX43 was abundantly expressed in young adult dental pulp, but expression of CX43 mRNA was dramatically decreased in aged human dental pulp. QRT-PCR analysis also showed the reduced expression of CX43 in aged pulp, and expression of CX43 in young pulp was significantly higher (about 10-fold, P < 0.01, Mann-Whitney U-test). CONCLUSION: Reduction of CX43 expression may be associated with the loss of viability in human dental pulp, and is considered as one characteristic of aged pulp.

Adolescent↗
PubMed2005

Reduction of osteocalcin expression in aged human dental pulp.

AIM: To investigate the expression of osteocalcin mRNA in young and in aged human dental pulp tissue to determine the characteristics of osteocalcin expression. METHODOLOGY: Human dental pulp tissues of the third molars were obtained from healthy young (17-23 years) and aged (>50 years) subjects, and total RNA was extracted. Osteocalcin mRNA expression was determined by RT-PCR and by quantitative real-time RT-PCR (QRT-PCR). The threshold cycle (Ct) value, which reflects the amount of PCR, was calculated and the difference between the value in young and aged pulp was statistically analysed. RESULTS: Osteocalcin mRNA was detected in all samples of human dental pulp tissue homogenates by RT-PCR analysis. Osteocalcin mRNA was expressed in young adult dental pulp but was decreased in aged human dental pulp. QRT-PCR analysis also showed a reduced expression of osteocalcin mRNA in aged human pulp. Expression of osteocalcin in young human pulp was significantly higher (about sixfold) than in aged pulp (P<0.01, Mann-Whitney U-test). CONCLUSION: Reduction of osteocalcin expression may be associated with the loss of viability in human dental pulp tissue, and may be a characteristic of aged human dental pulps.

Adolescent↗
PubMed2006

Quantitative determination of cucumber mosaic virus genome RNAs in virions by real-time reverse transcription-polymerase chain reaction.

A real-time RT-PCR procedure using the green fluorescent dye SYBR Green I was developed for determining the absolute and relative copies of cucumber mosaic virus (CMV) genomic RNAs contained in purified virions. Primers specific to each CMV ORF were designed and selected. Sequences were then amplified with length varying from 61 to 153 bp. Using dilution series of CMV genome RNAs prepared by in vitro transcription as the standard samples, a good linear correlation was observed between their threshold cycle (Ct) values and the logarithms of the initial template amounts. The copies of genomic RNA 1, RNA 2, RNA 3 and the subgenomic RNA 4 in CMV virions were quantified by this method, and the ratios were about 1.00:1.17:3.58:5.81. These results were confirmed by Lab-on-a-chip and northern blot hybridization assays. Our work is the first report concerning the relative amounts of different RNA fragments in CMV virions as a virus with tripartite genome.

Benzothiazoles↗
PubMed2004

Enrichment followed by quantitative PCR both for rapid detection and as a tool for quantitative risk assessment of food-borne thermotolerant campylobacters.

As part of a large international project for standardization of PCR (Food-PCR; www.pcr.dk), a multiplex, multiplatform, ready-to-go enrichment followed by a real-time PCR method, including an internal amplification control, was developed for detection of food-borne thermotolerant campylobacters in chickens. Chicken rinse samples were enriched in Bolton broth for 20 h, a simple and rapid (1-h) resin-based DNA extraction was performed, and DNA samples were then tested with two instrument platforms: ABI-PRISM 7700 and RotorGene 3000. The method was validated against an International Standard Organization (ISO)-based culture method by testing low, medium, and high levels of 12 spiked and 66 unspiked, presumably naturally contaminated, chicken rinse samples. In the RotorGene, a positive PCR response was detected in 40 samples of the 66. This was in complete agreement with the enriched ISO culture. The ABI-PRISM 7700 missed one culture-positive sample. Positive samples contained 10(2) to 10(7) CFU/ml after enrichment in Bolton broth. In the enriched samples a detection probability of 95% was obtained at levels of 1 x 10(3) and 2 x 10(3) CFU/ml in the RotorGene and ABI-PRISM, respectively. The amplification efficiency in both platforms was 90%, although the linear range of amplification of purified genomic DNA was 1.5 x 10(1) to 1 x 10(7) (R(2) = 1.00) for the RotorGene and 10(3) to 10(7) (R(2) = 0.99) for the ABI-PRISM. In RotorGene and ABI-PRISM the levels of precision of detection as determined by standard deviation (coefficients of variation) of 6-carboxyfluorescein (FAM) threshold cycle (Ct) values were 0.184 to 0.417 (0.65 to 2.57%) and 0.119 to 0.421 (0.59 to 1.82%), respectively. The results showed a correlation (R(2)) of 0.94 between the target FAM Ct values and CFU per milliliter of enriched naturally contaminated chicken samples, which indicates PCR's additional potential as a tool for quantitative risk assessment. Signal from the internal amplification control was detected in all culture-negative samples (VIC Ct: 23.1 to 28.1). The method will be taken further and validated in an international collaborative trial with regard to standardization.

Animals↗
PubMed2026

Improving isolate recovery and identification of the Shiga toxin type in Shiga toxin nucleic acid test-positive feces.

UNLABELLED: Infections caused by Shiga toxin-producing Escherichia coli (STEC) strains carrying Shiga toxin 2 (stx2) are more likely to result in severe complications; however, most nucleic acid amplification tests used for STEC diagnosis do not differentiate between stx1 and stx2. We therefore sought to optimize stx typing and isolate recovery methods to guide clinical and public health management. stx polymerase chain reaction (PCR)-positive feces were cultured using CHROMagar STEC and gram-negative broth, with Stx1 and/or Stx2 antigen detection by enzyme immunoassay (EIA) on colony growth or turbid broth. When cultures were EIA-negative, growth from MacConkey agar (MAC), Trypticase soy broth, and the gram-negative broth was then tested using a lab-developed typing PCR for stx1 and stx2. Colonies were isolated on CHROMagar STEC or MAC and identified using the typing PCR. Using both EIA and typing PCR, the stx types were identified in 96.0% of cases (381/397). In 65.2% (259/397) of cases, culture was EIA-positive, of which 34.0% (87/256) were Stx2-positive. Among cultures that were EIA-negative but typing PCR-positive, 64.8% (79/122) were stx2-positive (P < 0.0001 compared to EIA-positive). Using both EIA and typing PCR resulted in 72.6% (286/394) of cases with successful attempts at isolate recovery, compared to 60.7% (239/394) with EIA alone. E. coli O157 was recovered from more EIA-positive cases (19.3%, 46/239) than EIA-negative ones (4.3%, 2/47) (P = 0.0097). Typing PCR on cultures improves stx typing (particularly stx2) and isolate detection compared to EIA alone. Screening BD Max PCR and subsequent typing PCR results showed excellent concordance. IMPORTANCE: Escherichia coli strains with one or both types of Shiga toxins (stx1 and stx2) are a common cause of bacterial diarrhea and can lead to serious complications such as kidney failure, especially in children. Infection by stx2-positive strains is more likely to do so. Therefore, knowing whether the infection is caused by a strain carrying stx2 is important for risk assessment and case follow-up. The conventional way to diagnose these infections is to grow the bacteria from stool, but most laboratories currently use nucleic acid detection (e.g., bacterial DNA detection by polymerase chain reaction [PCR]), and these assays do not differentiate between the two toxin genes. Culture is therefore required to determine toxin type, as well as for public health outbreak investigations, which require an isolate for whole-genome sequencing for serotyping and cluster analysis. We identified culture media and a PCR-based method to detect stx2 in culture that improved the detection of stx2 and isolate recovery. Our findings provide more accurate results for clinicians to improve patient care and tools for public health teams to control and prevent outbreaks.

Humans↗
PubMed2002

Application of a 5' nuclease assay for detection of Lawsonia intracellularis in fecal samples from pigs.

A 5' nuclease assay was developed to detect Lawsonia intracellularis in porcine fecal samples. The specific probe and primers were chosen by using the 16S ribosomal DNA gene as a target. The 5' nuclease assay was used with a total of 204 clinical samples, and the results were compared to those of immunohistochemistry (IM) on ileal sections of the same animals. There was 91% agreement between the results of IM and the 5' nuclease assay. In the 5' nuclease assay, 111 (54%) of the pigs tested positive for L. intracellularis infection, with a mean cycle threshold (Ct) value of 27.2, whereas 98 (48%) of the pigs tested positive by IM. On average, the Ct and DeltaRn values for the positive samples were 27.2 (standard deviation [SD], 3.7) and 1.6 (SD, 0.7), respectively. A Ct value of 27.2 corresponds to a fecal excretion of approximately 10(7) L. intracellularis cells per g of feces. Furthermore, a total of 40 fecal samples derived from a herd known to be free from infection with L. intracellularis all tested negative, with a Ct value of 40. By using a Ct value of 36 as the cutoff limit, the detection limit of the assay was 1 L. intracellularis cell per PCR tube. In conclusion, the 5' nuclease assay that has been developed represents an applicable fast method for detection of L. intracellularis in fecal samples, with a sensitivity and specificity comparable to those of IM.

Animals↗
PubMed2003

Development of group- and serotype-specific one-step SYBR green I-based real-time reverse transcription-PCR assay for dengue virus.

A quantitative one-step SYBR Green I-based reverse transcription (RT)-PCR system was developed for the detection and differentiation of four different dengue virus serotypes in acute-phase serum samples. A set of group- and serotype-specific primer pairs was designed against conserved sequences in the core region and evaluated for clinical diagnosis. A linear relationship was obtained between the amount of input RNA and cycle threshold (Ct) value over a range of 10 to 10(7) PFU per ml of cell culture-derived dengue viruses. The detection limit of the group-specific primer pair was between 4.1 and 43.5 PFU/ml for four dengue serotypes. The detection limit of each of the serotype-specific primer pairs was calculated to be 10 PFU/ml for dengue virus serotype 1 (DEN-1), 4.6 PFU/ml for DEN-2, 4.1 PFU/ml for DEN-3, and 5 PFU/ml for DEN-4. Comparisons between the one-step SYBR Green-based RT-PCR assay and the conventional cell culture method in the clinical diagnosis of dengue virus infection from acute-phase serum samples of confirmed dengue patients were performed. The results showed that 83 and 67% of 193 acute-phase serum samples tested were positive by the one-step SYBR Green-based RT-PCR method and cell culture method, respectively. Further analysis showed that the one-step SYBR Green-based RT-PCR method could detect twice as many acute-phase serum samples with positive dengue-specific immunoglobulin M (IgM) and/or IgG antibodies than cell culture method. Our results demonstrate the potential clinical application of the one-step SYBR Green I-based RT-PCR assay for the detection and differentiation of dengue virus RNA.

Acute Disease↗
PubMed2025

Viral viability markers of SARS-CoV-2: a comparison of cell culture, genomic RNA RT-PCR, and subgenomic RNA RT-PCR.

UNLABELLED: Accurate methods to assess viral viability are crucial for determining isolation duration and antiviral therapy in immunocompromised patients. Although cell culture (CC) is the gold standard, it has limitations. Cycle threshold (Ct) values from genomic RNA (gRNA) RT-PCR and subgenomic RNA (sgRNA) RT-PCR have been proposed as markers of active viral replication. This study evaluated the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. This study aimed to evaluate the correlation between CC, gRNA Ct values, and sgRNA to identify the best viral viability marker. We conducted a prospective study on immunocompromised patients with suspected SARS-CoV-2 infection at a tertiary hospital (May 2021 to May 2023). Nasopharyngeal swabs were inoculated into Vero E6 cells and tested for gRNA using RT-PCR (Cobas 6800, Roche) and for sgRNA (non-commercial RT-PCR). The sensitivity (S), specificity (SP), positive (PPV) and negative predictive value (NPV), and accuracy were calculated using CC as the gold standard. Among 285 samples from 108 patients, gRNA RT-PCR had high S and NPV (1.0) but low SP (0.24) and moderate PPV (0.63). Ct analysis improved performance in extreme but not intermediate values. A Ct &#x2264; 30 maximized S but had low SP; Ct &#x2264; 25 yielded S (0.88), SP (0.89), PPV (0.92), NPV (0.84), and accuracy (0.88); sgRNA showed the highest S (0.99), SP (0.96), PPV (0.97), NPV (0.99), and accuracy (0.98). sgRNA detection is the best marker for identifying viable SARS-CoV-2, aiding decisions on isolation, antiviral treatment, or delaying chemotherapy in immunocompromised patients. IMPORTANCE: Identifying whether a patient still has contagious SARS-CoV-2 is essential for managing isolation, antiviral treatment, and other clinical decisions-especially in immunocompromised individuals. While viral culture is the gold standard for confirming viral viability, it is slow, expensive, and not widely available. Many hospitals rely on RT-PCR tests, but these detect viral genetic material whether or not the virus is still active. This study shows that detecting subgenomic RNA (sgRNA), a molecule only present when the virus is actively replicating, is a highly accurate way, a molecule only present when the virus is actively replicating, is a highly accurate way to determine whether the virus is still viable. Compared to standard PCR or viral culture, sgRNA testing better predicts who is truly infectious. These findings support sgRNA as a useful tool to guide clinical management and infection control in vulnerable patients.

Humans↗