Evaluation of Nucleic Acid Purification-Free One-Step Real-Time PCR Kit for Norovirus Infection

Abstract BACKGROUND Scrub typhus and severe fever with thrombocytopenia syndrome (SFTS) are the most common tick-borne illnesses in South Korea. Early differentiation of SFTS from scrub typhus in emergency departments is essential but difficult because of their overlapping epidemiology, shared risk factors, and similar clinical manifestations. METHODS We compared the diagnostic performance of one-step isothermal nucleic acid amplification with bio-optical sensor detection (iNAD) under isothermal conditions, which is rapid (20–30 min), with that of real-time PCR, in patients with a confirmed tick-borne illness. Fifteen patients with confirmed SFTS who provided a total of 15 initial blood samples and 5 follow-up blood samples, and 21 patients with confirmed scrub typhus, were evaluated. RESULTS The clinical sensitivity of iNAD (100%; 95% CI, 83–100) for SFTS was significantly higher than that of real-time PCR (75%; 95% CI, 51–91; P = 0.047), while its clinical specificity (86%; 95% CI, 65–97) was similar to that of real-time PCR (95%; 95% CI, 77–99; P = 0.61). The clinical sensitivity of iNAD for scrub typhus (100%; 95% CI, 81–100) was significantly higher than that of real-time PCR for scrub typhus (67%; 95% CI, 43–85; P = 0.009), while its clinical specificity (90%; 95% CI, 67–98) was similar to that of real-time PCR (95%; 95% CI, 73–100; P > 0.99). CONCLUSIONS iNAD is a valuable, rapid method of detecting SFTS virus and Orientia tsutsugamushi with high clinical sensitivity and specificity.

Download Full-text

Differing Effects of Standard and Harsh Nucleic Acid Extraction Procedures on Diagnostic Helminth Real-Time PCRs Applied to Human Stool Samples

Pathogens ◽

10.3390/pathogens10020188 ◽

2021 ◽

Vol 10 (2) ◽

pp. 188

Author(s):

Tanja Hoffmann ◽

Andreas Hahn ◽

Jaco J. Verweij ◽

Gérard Leboulle ◽

Olfert Landt ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Acid Extraction ◽

Nucleic Acid Extraction ◽

Pcr Assay ◽

Bead Beating ◽

Stool Samples ◽

Pcr Assays ◽

Human Stool

This study aimed to assess standard and harsher nucleic acid extraction schemes for diagnostic helminth real-time PCR approaches from stool samples. A standard procedure for nucleic acid extraction from stool and a procedure including bead-beating as well as proteinase K digestion were compared with group-, genus-, and species-specific real-time PCR assays targeting helminths and nonhelminth pathogens in human stool samples. From 25 different in-house and commercial helminth real-time PCR assays applied to 77 stool samples comprising 67 historic samples and 10 external quality assessment scheme samples positively tested for helminths, higher numbers of positive test results were observed after bead-beating-based nucleic acid extraction for 5/25 (20%) real-time PCR assays irrespective of specificity issues. Lower cycle threshold values were observed for one real-time PCR assay after the standard extraction scheme, and for four assays after the bead-beating-based scheme. Agreement between real-time PCR results after both nucleic acid extraction strategies according to Cohen’s kappa ranged from poor to almost perfect for the different assays. Varying agreement was observed in eight nonhelminth real-time PCR assays applied to 67 historic stool samples. The study indicates highly variable effects of harsh nucleic acid extraction approaches depending on the real-time PCR assay used.

Download Full-text

Identifying the etiologic role of Parvovirus B19 in non-immune hydrops fetalis by histopathology, immunohistochemistry and nucleic acid testing: a retrospective study

Open Medicine ◽

10.2478/s11536-007-0029-z ◽

2007 ◽

Vol 2 (3) ◽

pp. 271-279 ◽

Cited By ~ 1

Author(s):

Koray Ergunay ◽

Gulcin Altinok ◽

Bora Gurel ◽

Ahmet Pinar ◽

Arzu Sungur ◽

...

Keyword(s):

Nucleic Acid ◽

Real Time ◽

San Francisco ◽

Real Time Pcr ◽

Nested Pcr ◽

Parvovirus B19 ◽

Etiologic Agent ◽

Hydrops Fetalis ◽

Nucleic Acid Detection

AbstractIntrauterine Parvovirus B19 infections may cause fetal anemia, non-immune hydrops fetalis or abortion. This study focuses on the pathogenic role of Parvovirus B19 in non-immune hydrops fetalis at Hacettepe University, a major reference hospital in Turkey. Twenty-two cases of non-immune hydrops fetalis were retrospectively selected out of a total of 431 hydrops fetalis specimens from the Department of Pathology archieves. Paraffine embedded tissue sections from placental and liver tissues from each case were evaluated by histopathology, immunohistochemistry, nested PCR and commercial quantitative Real-time PCR. Viral DNA was detected in placental tissues by Real-time PCR in 2 cases (2/22, 9.1%) where histopathology also revealed changes suggestive of Parvovirus B19 infection. No significant histopathologic changes were observed for the remaining sections. Nested PCR that targets the VP1 region of the viral genome and immunohistochemistry for viral capsid antigens were negative for all cases. As a result, Parvovirus B19 is identified as the etiologic agent for the development of non-immune hydrops fetalis for 9.1% of the cases in Hacettepe University, Turkey. Real-time PCR is observed to be an effective diagnostic tool for nucleic acid detection from paraffine embedded tissues. Part of this study was presented as a poster at XIIIth International Congress of Virology, San Francisco, USA (Abstract V-572).

Download Full-text

MGB-based one-step multiplex real-time PCR method for rapid detection of HPV

Cancer Biomarkers ◽

10.3233/cbm-130298 ◽

2013 ◽

Vol 12 (3) ◽

pp. 107-113 ◽

Cited By ~ 1

Author(s):

Jie Huang ◽

Chun Gao ◽

Xilai Ding ◽

Shoufang Qu ◽

Licheng Liu ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

One Step ◽

Pcr Method

Download Full-text

Locked Nucleic Acid Probe-Based Real-Time PCR Assay for the Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis

PLoS ONE ◽

10.1371/journal.pone.0143444 ◽

2015 ◽

Vol 10 (11) ◽

pp. e0143444 ◽

Cited By ~ 6

Author(s):

Yong Zhao ◽

Guilian Li ◽

Chongyun Sun ◽

Chao Li ◽

Xiaochen Wang ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Locked Nucleic Acid ◽

Pcr Assay ◽

Locked Nucleic Acid Probe

Download Full-text

P1.09 Diagnosingmycoplasma genitalium: comparison of four nucleic acid amplification tests and use of the speedx real-time pcr assay for azithromycin resistance

10.1136/sextrans-2017-053264.117 ◽

2017 ◽

Author(s):

Charlotte Gaydos ◽

Justin Hardick ◽

Susan Tuddenham ◽

Maria Trent

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Real Time Pcr ◽

Nucleic Acid Amplification ◽

Pcr Assay ◽

Nucleic Acid Amplification Tests ◽

Azithromycin Resistance

Download Full-text

Molecular Detection of Adenovirus in Tap Water from Coastal Areas Of Karachi Pakistan:The Real-Time PCR Assay

American Journal of Clinical Pathology ◽

10.1093/ajcp/aqab191.299 ◽

2021 ◽

Vol 156 (Supplement_1) ◽

pp. S140-S140

Author(s):

A Kalam

Keyword(s):

Nucleic Acid ◽

Real Time ◽

Low Income ◽

Water Samples ◽

Real Time Pcr ◽

Tap Water ◽

Watery Diarrhea ◽

Acid Extraction ◽

Pcr Assay ◽

Viral Contamination

Abstract Introduction/Objective Diarrhea is a major source of morbidity and mortality in low-income and middle-income countries. In underdeveloped countries, diseases caused by viruses identified in environmental samples cause major health problems. Little knowledge about the frequency and pattern of viral contamination of drinking water sources in these resource-poor settings. Adenovirus which causes watery diarrhea, particular has been recognized as important causal pathogen. Adenovirus remains a global threat to public health and an indicator of inequity and lack of social development. Tap water samples from coastal sites in Karachi between 2019 and 2020 over a period of 11 months. The total of 40 tap water sample was examined for infectious Adenovirus by a real time polymerase chain reaction (PCR) amplification. Methods/Case Report This Pilot study is conducted on tap water samples from Karachi Pakistan, n=40 are processed. Extraction of nucleic acid from all filtered water samples collected with Sterivex filter units by using Qiagen DNeasy Power Water Sterivex Kit. As per the manufacturer’s instruction. Phocine herpesvirus(PhHV) is added as an external positive control to monitor the efficiency of nucleic acid extraction and amplification. TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) is being used in probe based real time PCR assay,the below 35 Ct value is considered as a positive sample. Results (if a Case Study enter NA) Results showed the total of 37.7% of the sources were positive for adenovirus.The level of viral contamination was moderate to high. Conclusion The results has been showed that no seasonal pattern for viral contaminations was found after samples obtained during the dry and wet seasons were compared. Further the Real time PCR assay increases the sensitivity and provides the high resolution of pathogen detection.

Download Full-text