Development of a quantitative real-time PCR assay for sapovirus in children under 5-years-old in Regina Margherita Hospital of Turin, Italy

2017 ◽  
Vol 63 (4) ◽  
pp. 296-302 ◽  
Author(s):  
Massimiliano Bergallo ◽  
Ilaria Galliano ◽  
Paola Montanari ◽  
Martina Rosa Brusin ◽  
Serena Finotti ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Acute Gastroenteritis ◽  
Taqman Assay ◽  
Quantitative Detection ◽  
Common Disease ◽  
Median Viral Load ◽  
Significant Difference ◽  
Pcr Method ◽  
Children Under 5

Gastroenteritis is a common disease in children. It is characterized by diarrhea, vomiting, abdominal pain, and fever. Sapovirus (SaV) is a causative agent of acute gastroenteritis, but it causes milder illness than do rotavirus and norovirus. There is high variability in the analytical performance of quantitative PCR-based assays among clinical laboratories. This study developed a reverse transcription real-time PCR method to detect SaV in fecal specimens collected from children under 5-years-old with acute gastroenteritis. Of 137 episodes of acute gastroenteritis, 15 (10.9%) were associated with SaV genomic detection, with a median viral load of 6.6(log10) ± 7.1(log10) genomes/mg fecal specimens. There was a significant difference in detection rate between males and females (9.48% (13/15) vs. 1.46% (2/15), p = 0.0232). Among the 15 SaV-positive cases, 6 were also positive for rotavirus. Viral RNA recovery rate ranged from 46% to 77% in the manual RNAzol protocol and from 31% to 90% in the automated Maxwell protocol. We also studied whether human genomic DNA influences the sensitivity of the assay: its presence caused a decrease in PCR sensitivity. The development of a laboratory-designed real-time PCR TaqMan assay for quantitative detection of SaV and the optimization and standardization of this assay, using stools of children with acute gastroenteritis, are described.

scholarly journals Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

2021 ◽  
Author(s):  
Stephen Tukwasibwe ◽  
James A. Traherne ◽  
Olympe Chazara ◽  
Jyothi Jayaraman ◽  
John Trowsdale ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Malaria Transmission ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Association Studies ◽  
Transmission Intensity ◽  
Malaria Transmission Intensity ◽  
Significant Difference ◽  
Pcr Method

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches.Methods: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method developed will be useful in disease association studies involving large cohorts.

Quantitative Real-Time Polymerase Chain Reaction Detection of BK Virus Using Labeled Primers

2010 ◽  
Vol 134 (3) ◽  
pp. 444-448 ◽  
Author(s):  
Zhengming Gu ◽  
Jianmin Pan ◽  
Matthew J. Bankowski ◽  
Randall T. Hayden
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Polymerase Chain Reaction ◽  
Bk Virus ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Pcr Method ◽  
Polymerase Chain

Abstract Context.—BK virus infections among immunocompromised patients are associated with disease of the kidney or urinary bladder. High viral loads, determined by quantitative polymerase chain reaction (PCR), have been correlated with clinical disease. Objective.—To develop and evaluate a novel method for real-time PCR detection and quantification of BK virus using labeled primers. Design.—Patient specimens (n = 54) included 17 plasma, 12 whole blood, and 25 urine samples. DNA was extracted using the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche Applied Science, Indianapolis, Indiana); sample eluate was PCR-amplified using the labeled primer PCR method. Results were compared with those of a user-developed quantitative real-time PCR method (fluorescence resonance energy transfer probe hybridization). Results.—Labeled primer PCR detected less than 10 copies per reaction and showed quantitative linearity from 101 to 107 copies per reaction. Analytical specificity of labeled primer PCR was 100%. With clinical samples, labeled primer PCR demonstrated a trend toward improved sensitivity compared with the reference method. Quantitative assay comparison showed an R2 value of 0.96 between the 2 assays. Conclusions.—Real-time PCR using labeled primers is highly sensitive and specific for the quantitative detection of BK virus from a variety of clinical specimens. These data demonstrate the applicability of labeled primer PCR for quantitative viral detection and offer a simplified method that removes the need for separate oligonucleotide probes.

Quantitative Detection of Campylobacter jejuni on Fresh Chicken Carcasses by Real-Time PCR

2007 ◽  
Vol 70 (6) ◽  
pp. 1373-1378 ◽  
Author(s):  
ANNA-CLARA RÖNNER ◽  
HANS LINDMARK
Keyword(s):  
Detection Limit ◽  
Real Time ◽  
Dna Extraction ◽  
Campylobacter Jejuni ◽  
Real Time Pcr ◽  
Close Correlation ◽  
Quantitative Detection ◽  
Quantitative Real Time Pcr ◽  
Direct Plating ◽  
Pcr Method

Campylobacter jejuni infection is a significant cause of foodborne gastroenteritis worldwide. Consumption and handling of poultry products is believed to be the primary risk factor for campylobacteriosis. Risk assessments require quantitative data, and C. jejuni is enumerated usually by direct plating, which sometimes allows growth of non-Campylobacter bacteria. The objective of the present study was to develop a quantitative real-time PCR method (q-PCR) for enumerating C. jejuni in chicken rinse without a culturing step. The procedure to obtain the template for the PCR assay involved (i) filtration of 10 ml of chicken rinse, (ii) centrifugation of the sample, and (iii) total DNA extraction from the pellet obtained using a commercial DNA extraction kit. The detection limit of the method was comparable to that for plating 100 μl of chicken rinse on modified charcoal cefoperazone deoxycholate agar, and the detection limit could be further improved 10-fold by concentrating the DNA eluate by ethanol precipitation. A close correlation for spiked chicken rinse was obtained for the results of the quantitative real-time PCR method and direct plating (r = 0.99). The coefficient of correlation for the methods was 0.87 when samples from chicken carcasses on the slaughter line were analyzed, whereas a lower correlation (r = 0.76) was obtained when samples from retail carcasses were analyzed. Greater variation in the proportion of dead and/or viable but not culturable Campylobacter types in the retail samples may explain the decreased correlation between the methods. Overall, the new method is simple and fast and the results obtained are closely correlated with those for direct plating for samples containing a low proportion of dead Campylobacter cells.

scholarly journals Molecular Study of Norovirus in Pediatric Patients with Gastroenteritis

2019 ◽  
Vol 13 (1) ◽  
pp. 324-329
Author(s):  
Maysaa El Sayed Zaki ◽  
Abdel-Rahaman Eid ◽  
Amany Y. El Ashry ◽  
Nashwa M. Al-Kasaby
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Acute Gastroenteritis ◽  
Predictive Value ◽  
Pediatric Patients ◽  
Virus Detection ◽  
Molecular Study ◽  
Antigen Detection ◽  
Cross Sectional ◽  
Significant Difference

Aim: The aim of the present study was to detect the prevalence of norovirus and genotypes determination by real-time PCR among children below 18 years as an etiology of acute gastroenteritis and to compare rapid detection of norovirus by Enzyme-Linked Immunoassay (ELISA) to virus detection by real-time PCR. Methods: The research was a cross-sectional study conducted on children below 18 years complaining of community-acquired acute gastroenteritis. A stool sample was subjected to direct-antigen detection by ELISA for norovirus and molecular study by real-time polymerase chain reaction. Results: The study included 200 children with acute gastroenteritis with a mean age of 6.7±3.8 years. Norovirus antigen was detected by EIA in 34.5% and by real-time PCR in 30.5% of studied children with genotype GII, the predominant detected genotype (80.97%). Both real-time PCR and antigen detection of norovirus were positive in 43 (70.5%) of the children and negative in 113(81.3%) of the studied children. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy for antigen detection by ELISA were 70.5%, 81.3%, 62.3%, 86.3% and 78%, respectively. Comparison between patients positive for norovirus and those negative for norovirus by real-time PCR revealed non-significant difference as regards age, sex, the season of occurrence and residence. Conclusion: The present study highlights that norovirus prevalence is common among pediatric patients with gastroenteritis above 5 years with GII genotype as the prevalent genotype. There was a significant correlation between positive and negative results of antigen detection of norovirus by ELISA and detection of RNA of norovirus by real-time PCR in stool samples. However, the screening for norovirus by ELISA has limited sensitivity and needs to be associated with a molecular method for accurate diagnosis of sporadic cases of gastroenteritis.

scholarly journals Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

2020 ◽  
Author(s):  
Stephen Tukwasibwe ◽  
James A. Traherne ◽  
Olympe Chazara ◽  
Jyothi Jayaraman ◽  
John Trowsdale ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Malaria Transmission ◽  
Real Time ◽  
High Throughput ◽  
Real Time Pcr ◽  
Association Studies ◽  
Transmission Intensity ◽  
Malaria Transmission Intensity ◽  
Significant Difference ◽  
Pcr Method

Abstract Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. Our aim was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches. Methods: We used high throughput multiplex quantitative real-time PCR method to genotype KIR genetic variants and copy number variation and developed a high-throughput real-time PCR method to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1 , KIR2DL5, KIR2DS5 and KIR2DS1 genes are potentially beneficial in malaria as these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method we have developed will be useful in disease association studies involving large cohorts.

scholarly journals Development of a mreB-targeted real-time PCR method for the quantitative detection of Vibrio harveyi in seawater and biofilm from aquaculture systems

Aquaculture ◽  
2020 ◽  
Vol 525 ◽  
pp. 735337 ◽  
Author(s):  
Julia Mougin ◽  
Roxane Roquigny ◽  
Marie-Agnès Travers ◽  
Thierry Grard ◽  
Maryse Bonnin-Jusserand ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Harveyi ◽  
Quantitative Detection ◽  
Pcr Method

scholarly journals Multiplex Fast Real-Time PCR for Quantitative Detection and Identification of cos- and pac-Type Streptococcus thermophilus Bacteriophages

2008 ◽  
Vol 74 (15) ◽  
pp. 4779-4781 ◽  
Author(s):  
Beatriz del Rio ◽  
María Cruz Martín ◽  
Noelia Martínez ◽  
Alfonso H. Magadán ◽  
Miguel A. Alvarez
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Industrial Process ◽  
Streptococcus Thermophilus ◽  
Quantitative Detection ◽  
Detection And Identification ◽  
Pcr Method

ABSTRACT The fermentation of milk by Streptococcus thermophilus is a widespread industrial process that is susceptible to bacteriophage attack. In this work, a preventive fast real-time PCR method for the detection, quantification, and identification of types of S. thermophilus phages in 30 min is described.

Preliminary Evaluation of Flow-Through Immunocapture followed by Real-Time PCR for the Detection of Salmonella Serovars on Tomato Surfaces within 8 Hours

2006 ◽  
Vol 69 (9) ◽  
pp. 2253-2257 ◽  
Author(s):  
HYUN-GYUN YUK ◽  
BENJAMIN R. WARREN ◽  
KEITH R. SCHNEIDER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Preliminary Evaluation ◽  
Conventional Culture ◽  
Significant Difference ◽  
Assay Time ◽  
Flow Through ◽  
Pcr Method ◽  
Salmonella Serovars

This study reports a preliminary evaluation of flow-through immunocapture (FTI) followed by real-time PCR (FTI-PCR) for the detection of Salmonella serovars on tomato surfaces within 8 h. The FTI-PCR method was compared with real-time PCR, direct plating of FTI beads on xylose lysine desoxycholate (XLD), and the conventional culture method for Salmonella found in the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM). Unwaxed green tomatoes were spot inoculated with a five-serovar Salmonella cocktail on smooth surfaces at levels of 100 to 104 CFU per tomato and washed in lactose broth (LB) using a shake-rub method. The resulting LB rinse was incubated at 37°C for 4 h prior to analysis by FTI-XLD, real-time PCR, or FTI-PCR and for 24 h as the first step in the BAM Salmonella culture method. For FTI-XLD, the observed lowest detection level (LDL) was 4.6 × 101 CFU per tomato. There was no significant difference in performance between the FTI-XLD method and the BAM Salmonella culture method (P &gt; 0.05); however, the FTI-XLD method reduced the overall assay time by 48 h. For real-time PCR and FTI-PCR, the observed LDLs were 4.6 × 101 and 9.2 × 100 CFU per tomato, respectively. The FTI-PCR method was superior to the BAM Salmonella culture method (P &lt; 0.05) for the detection of Salmonella serovars on tomato surfaces and was completed within 8 h.

Sign in / Sign up

Export Citation Format

Share Document