scholarly journals Reverse transcription quantitative PCR to detect low density malaria infections

2020 ◽  
Author(s):  
Peter Christensen ◽  
Zbynek Bozdech ◽  
Wanitda Watthanaworawit ◽  
Laurent Renia ◽  
Benoit Malleret ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Malaria Elimination ◽  
Malaria Diagnosis ◽  
Sample Volume ◽  
Malaria Prevalence ◽  
Low Density ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Increased Sensitivity

AbstractTargeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method. Here we evaluated a real-time quantitative reverse transcription PCR (qRT-PCR) method that significantly reduces the required sample volume compared to uPCR. To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were compared using uPCR and qRT-PCR. Plasmodium spp. qRT-PCR confirmed 18 of 21 uPCR Plasmodium falciparum positives, while P. falciparum specific qRT-PCR confirmed 17 of the 21 uPCR P. falciparum positives. Combining both qRT-PCR results increased the sensitivity to 100% and specificity was 95.1%. These results show that malaria detection in areas of low transmission and LDMI, can benefit from the increased sensitivity of qRT-PCR especially where sample volume is limited.

scholarly journals Reverse transcription PCR to detect low density malaria infections

2021 ◽  
Vol 6 ◽  
pp. 39
Author(s):  
Peter Christensen ◽  
Zbynek Bozdech ◽  
Wanitda Watthanaworawit ◽  
Laurent Renia ◽  
Benoit Malleret ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Malaria Elimination ◽  
Malaria Diagnosis ◽  
Sample Volume ◽  
Malaria Prevalence ◽  
Low Density ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Increased Sensitivity ◽  
Downstream Analysis

Background: Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method. Methods: Here we evaluated a real-time reverse transcription PCR (RT-PCR) method that reduces the required sample volume compared to uPCR. To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were compared using uPCR and RT-PCR. Results: Plasmodium spp. RT-PCR confirmed 18 of 21 uPCR Plasmodium falciparum positives, while P. falciparum specific RT-PCR confirmed 17 of the 21 uPCR P. falciparum positives. Combining both RT-PCR results increased the sensitivity to 100% and specificity was 95.1%. Conclusion: Malaria detection in areas of low transmission and LDMI can benefit from the increased sensitivity of ribosomal RNA detection by RT-PCR, especially where sample volume is limited. Isolation of high quality RNA also allows for downstream analysis of malaria transcripts.

scholarly journals Reverse transcription PCR to detect low density malaria infections

2021 ◽  
Vol 6 ◽  
pp. 39
Author(s):  
Peter Christensen ◽  
Zbynek Bozdech ◽  
Wanitda Watthanaworawit ◽  
Laurent Rénia ◽  
Benoît Malleret ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Malaria Elimination ◽  
Malaria Diagnosis ◽  
Sample Volume ◽  
Malaria Prevalence ◽  
Low Density ◽  
Reverse Transcription Pcr ◽  
Detection Program ◽  
Increased Sensitivity ◽  
Downstream Analysis

Background: Targeted malaria elimination strategies require highly sensitive tests to detect low density malaria infections (LDMI). Commonly used methods for malaria diagnosis such as light microscopy and antigen-based rapid diagnostic tests (RDTs) are not sensitive enough for reliable identification of infections with parasitaemia below 200 parasites per milliliter of blood. While targeted malaria elimination efforts on the Thailand-Myanmar border have successfully used high sample volume ultrasensitive quantitative PCR (uPCR) to determine malaria prevalence, the necessity for venous collection and processing of large quantities of patient blood limits the widespread tractability of this method. Methods: Here we evaluated a real-time reverse transcription PCR (RT-qPCR) method that reduces the required sample volume compared to uPCR. To do this, 304 samples collected from an active case detection program in Kayin state, Myanmar were compared using uPCR and RT-qPCR. Results: Plasmodium spp. RT-qPCR confirmed 18 of 21 uPCR Plasmodium falciparum positives, while P. falciparum specific RT-qPCR confirmed 17 of the 21 uPCR P. falciparum positives. Combining both RT-qPCR results increased the sensitivity to 100% and specificity was 95.1%. Conclusion: Malaria detection in areas of low transmission and LDMI can benefit from the increased sensitivity of ribosomal RNA detection by RT-PCR, especially where sample volume is limited. Isolation of high quality RNA also allows for downstream analysis of malaria transcripts.

scholarly journals Application of a Receptor-Binding Capture Quantitative Reverse Transcription-PCR Assay To Concentrate Human Norovirus from Sewage and To Study the Distribution and Stability of the Virus

2011 ◽  
Vol 78 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Peng Tian ◽  
David Yang ◽  
Liangwen Pan ◽  
Robert Mandrell
Keyword(s):  
Receptor Binding ◽  
Reverse Transcription ◽  
Magnetic Beads ◽  
Sample Volume ◽  
Sewage Effluent ◽  
Human Norovirus ◽  
Qrt Pcr ◽  
Quantitative Reverse Transcription Pcr ◽  
Reverse Transcription Pcr ◽  
Charged Membrane

ABSTRACTWater is an important route for human norovirus (HuNoV) transmission. Using magnetic beads conjugated with blood group-like antigens (HuNoV receptors), we developed a simple and rapid receptor-binding capture and magnetic sequestration (RBCMS) method and compared it to the existing negatively charged membrane absorption/elution (NCMAE) method for concentrating HuNoV from sewage effluent. RBCMS required 6-fold-less sample volume than the NCMAE method and also resulted in a significantly higher yield of HuNoV. The NCMAE and RBCMS concentrations of genogroup I (GI) HuNoV measured by quantitative reverse transcription-PCR (qRT-PCR) resulted in average threshold cycle (CT) values of 34.68 (8.68 copies, 252-fold concentration) versus 34.07 (13.05 copies, 477-fold concentration), respectively; the NCMAE and RBCMS concentrations of genogroup II (GII) HuNoV were measured as averageCTvalues of 33.32 (24.7 copies, 239-fold concentration) versus 32.38 (46.9 copies, 333-fold concentration), respectively. The specificity of qRT-PCR was confirmed by traditional RT-PCR and an RNase I protection assay. The qRT-PCR signal from RBCMS-concentrated HuNoV treated with RNase I indicated that it was from encapsidated RNA and, probably, viable virus. In contrast, the qRT-PCR signal from NCMAE-concentrated HuNoV was not protected from RNase I and, likely, degradation. Both GI and GII HuNoV were detected from sewage effluent samples collected between April and July with average concentrations of 7.8 × 103genomic copies per liter (gc/liter) and 4.3 × 104gc/liter, respectively. No GI and <2% GII HuNoV were detected in sewage samples stored at room temperature for 4 weeks. We conclude that RBCMS requires less sample volume, has better recovery and sensitivity, and is faster than NCMAE for detection of HuNoV in sewage.

An Eco-friendly Fabrication of Silver Chloride Nanoparticles (AgCLNPs) using Onopordum acanthium L extract Induces Apoptosis in Breast Cancer MDA-MB-232 Cells

2021 ◽  
Author(s):  
Seyedeh Negin Shahcheraghi ◽  
Seyed Ataollah Sadat Shandiz ◽  
Bahareh Pakpour
Keyword(s):  
Breast Cancer ◽  
Real Time ◽  
Cell Lines ◽  
Reverse Transcription ◽  
Hek293 Cell ◽  
Silver Chloride ◽  
Hek293 Cells ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Onopordum Acanthium

Abstract In the current experimental work, silver chloride nanoparticles (AgClNPs) were fabricated using Onopordum acanthium L extract and their apoptotic and cytotoxicity properties on breast cancer MDA_MB232 and normal HEK293 cell lines were also evaluated. AgClNPs formation was determined by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) profile. Effect of fabricated AgClNPs on MDA_MB232 and HEK293 cells viability was performed using colorimetric MTT assay. Alterations in the mRNA expression levels of CAD and Bax genes in MDA-MB-232 cells were done using quantitative real-time reverse transcription-PCR (qRT-PCR) method. Subsequently, apoptotic properties were determined using flow cytometry and fluorescence microscopy studies. MTT results investigated that AgCLNPs have a significant dose-dependent lethal activity on MDA_MB232 compared to HEK293 cell lines. Quantitative real-time reverse transcription-PCR (qRT-PCR) results have also shown that AgCLNPs could up-regulate the apoptotic Bax and CAD gene expressions in the MDA_MB232 cells. Additionally, apoptotic assessment was performed by cell cycle analysis, annexin V/PI test, Hoescht 33258 dye, acridine orange and ethidium bromide (AO/EB) staining along with the detection of the reactive oxygen species (ROS) generation. Our results suggest that novel silver chloride nanoparticles fabricated by Onopordum acanthium L extract can display some promising cytotoxic properties through inducing apoptosis pathway.

scholarly journals NewIn SituCapture Quantitative (Real-Time) Reverse Transcription-PCR Method as an Alternative Approach for Determining Inactivation of Tulane Virus

2014 ◽  
Vol 80 (7) ◽  
pp. 2120-2124 ◽  
Author(s):  
Dapeng Wang ◽  
Shuxia Xu ◽  
David Yang ◽  
Glenn M. Young ◽  
Peng Tian
Keyword(s):  
Tissue Culture ◽  
Reverse Transcription ◽  
Viral Genome ◽  
Viral Infectivity ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Alternative Approach ◽  
Tulane Virus ◽  
Pcr Method

ABSTRACTHuman noroviruses (HuNoVs) are the major cause of epidemic nonbacterial gastroenteritis. Although quantitative (real-time) reverse transcription-PCR (qRT-PCR) is widely used for detecting HuNoVs, it only detects the presence of viral RNA and does not indicate viral infectivity. Human blood group antigens (HBGAs) have been identified as receptors/co-receptors for both HuNoVs and Tulane virus (TV) and are crucial for viral infection. We propose that viral infectivity can be evaluated with a molecular assay based on receptor-captured viruses. In this study, we employed TV as an HuNoV surrogate to validate the HBGA-based capture qRT-PCR method against the 50% tissue culture infectious dose (TCID50) method. We employed type B HBGA on an immuno-well module to concentrate TV, followed by amplification of the captured viral genome byin situqRT-PCR. We first demonstrated that thisin situcapture qRT-PCR (ISC-qRT-PCR) method could effectively concentrate and detect TV. We then treated TV under either partial or full inactivation conditions and measured the remaining infectivity by ISC-qRT-PCR and a tissue culture-based amplification method (TCID50). We found that the ISC-qRT-PCR method could be used to evaluate virus inactivation deriving from damage to the capsid and study interactions between the capsid and viral receptor. Heat, chlorine, and ethanol treatment primarily affect the capsid structure, which in turns affects the ability of the capsid to bind to viral receptors. Inactivation of the virus by these methods could be reflected by the ISC-qRT-PCR method and confirmed by TCID50assay. However, the loss of the infectivity caused by damage to the viral genome (such as that from UV irradiation) could not be effectively reflected by this method. Despite this limitation, the ISC-qRT-PCR provides an alternative approach to determine inactivation of Tulane virus. A particular advantage of the ISC-qRT-PCR method is that it is also a faster and easier method to effectively recover and detect the viruses, as there is no need to extract viral RNA or to transfer the captured virus from magnetic beads to PCR tubes for further amplification. Therefore, ISC-qRT-PCR can be easily adapted for use in automated systems for multiple samples.

scholarly journals Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis

2005 ◽  
Vol 109 (4) ◽  
pp. 365-379 ◽  
Author(s):  
Stephen A. Bustin ◽  
Reinhold Mueller
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Therapy Monitoring ◽  
Clinical Diagnostics ◽  
Clinical Diagnostic Laboratory ◽  
Diagnostic Laboratory ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Clinical Diagnostic ◽  
Pcr Assays

qRT-PCR (real-time reverse transcription-PCR) has become the benchmark for the detection and quantification of RNA targets and is being utilized increasingly in novel clinical diagnostic assays. Quantitative results obtained by this technology are not only more informative than qualitative data, but simplify assay standardization and quality management. qRT-PCR assays are most established for the detection of viral load and therapy monitoring, and the development of SARS (severe acute respiratory syndrome)-associated coronavirus qRT-PCR assays provide a textbook example of the value of this technology for clinical diagnostics. The widespread use of qRT-PCR assays for diagnosis and the detection of disease-specific prognostic markers in leukaemia patients provide further examples of their usefulness. Their value for the detection of disease-associated mRNA expressed by circulating tumour cells in patients with solid malignancies is far less apparent, and the clinical significance of results obtained from such tests remains unclear. This is because of conceptual reservations as well as technical limitations that can interfere with the diagnostic specificity of qRT-PCR assays. Therefore, although it is evident that qRT-PCR assay has become a useful and important technology in the clinical diagnostic laboratory, it must be used appropriately and it is essential to be aware of its limitations if it is to fulfil its potential.

scholarly journals Comparison of Automated Quantitative Reverse Transcription-PCR and Direct Fluorescent-Antibody Detection for Routine Rabies Diagnosis in the United States

2015 ◽  
Vol 53 (9) ◽  
pp. 2983-2989 ◽  
Author(s):  
Michelle Dupuis ◽  
Scott Brunt ◽  
Kim Appler ◽  
April Davis ◽  
Robert Rudd
Keyword(s):  
United States ◽  
Reverse Transcription ◽  
Gold Standard ◽  
Fluorescent Antibody ◽  
The United States ◽  
Pcr Assay ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Direct Fluorescent Antibody ◽  
Rabies Diagnosis

Rabies virus found worldwide and prevalent throughout the United States continues to be a public health concern. Direct-fluorescent antibody (DFA) detection remains the gold standard for rabies virus diagnostics. Assessing the utility of a high-throughput molecular platform such as the QIAsymphony SP/AS, in conjunction with quantitative reverse transcription-PCR (qRT-PCR), to augment or potentially replace the DFA test, was the focus of this project. Here we describe a triplex qRT-PCR assay, including assembly and evaluation for sensitivity, specificity, and ability to detect variants. Additionally, we compared the qRT-PCR assay to the gold standard direct fluorescent-antibody test. More than 1,000 specimens submitted for routine rabies diagnosis were tested to directly compare the two methods. All results were in agreement between the two methods, with one additional specimen detected by qRT-PCR below the limits of the DFA sensitivity. With the proper continued validation for variant detection, molecular methods have a place in routine rabies diagnostics within the United States.

scholarly journals Rapid Detection of Novel Coronavirus (COVID-19) by Reverse Transcription-Loop-Mediated Isothermal Amplification

2020 ◽  
Author(s):  
Laura E. Lamb ◽  
Sarah N. Bartolone ◽  
Elijah Ward ◽  
Michael B. Chancellor
Keyword(s):  
Diagnostic Test ◽  
Reverse Transcription ◽  
Simulated Patient ◽  
Isothermal Amplification ◽  
Rapid Screening ◽  
Health Concern ◽  
Loop Mediated Isothermal Amplification ◽  
Qrt Pcr ◽  
Reverse Transcription Pcr ◽  
Novel Coronavirus

AbstractNovel Corona virus (COVID-19 or 2019-nCoV) is an emerging global health concern that requires a rapid diagnostic test. Quantitative reverse transcription PCR (qRT-PCR) is currently the standard for COVID-19 detection; however, Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) may allow for faster and cheaper field based testing at point-of-risk. The objective of this study was to develop a rapid screening diagnostic test that could be completed in under 30 minutes. Simulated patient samples were generated by spiking serum, urine, saliva, oropharyngeal swabs, and nasopharyngeal swabs with a portion of the COVID-19 nucleic sequence. The samples were tested using RT-LAMP as well as by conventional qRT-PCR. Specificity of the RT-LAMP was evaluated by also testing against other related coronaviruses. RT-LAMP specifically detected COVID-19 in simulated patient samples. This test was performed in under 30 minutes. This approach could be used for monitoring of exposed individuals or potentially aid with screening efforts in the field and potential ports of entry.

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