scholarly journals Real-time digital polymerase chain reaction (PCR) as a novel technology improves limit of detection for rare allele assays

2021 ◽  
Vol 0 (0) ◽  
pp. 0-0
Author(s):  
Jiachen Xu ◽  
Kyra Duong ◽  
Zhenlin Yang ◽  
Kavanaugh Kaji ◽  
Jiajia Ou ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Rare Allele ◽  
Chain Reaction ◽  
Novel Technology ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

scholarly journals Digital Droplet PCR for SARS-CoV-2 Resolves Borderline Cases

2021 ◽  
Author(s):  
Jing Xu ◽  
Timothy Kirtek ◽  
Yan Xu ◽  
Hui Zheng ◽  
Huiyu Yao ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
False Negative ◽  
Accurate Method ◽  
Viral Envelope ◽  
Chain Reaction ◽  
Borderline Cases ◽  
Droplet Pcr ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Abstract Objectives The Bio-Rad SARS-CoV-2 ddPCR Kit (Bio-Rad Laboratories) was the first droplet digital polymerase chain reaction (ddPCR) assay to receive Food and Drug Administration (FDA) Emergency Use Authorization approval, but it has not been evaluated clinically. We describe the performance of ddPCR—in particular, its ability to confirm weak-positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) results. Methods We clinically validated the Bio-Rad Triplex Probe ddPCR Assay. The limit of detection was determined by using serial dilutions of SARS-CoV-2 RNA in an artificial viral envelope. The ddPCR assay was performed according to the manufacturer’s specifications on specimens confirmed to be positive (n = 48) or negative (n = 30) by an FDA-validated reverse transcription–polymerase chain reaction assay on the m2000 RealTime system (Abbott). Ten borderline positive cases were also evaluated. Results The limit of detection was 50 copies/mL (19 of 20 positive). Forty-seven specimens spanning a range of quantification cycles (2.9-25.9 cycle numbers) were positive by this assay (47 of 48; 97.9% positive precent agreement), and 30 negative samples were confirmed as negative (30 of 30; 100% negative percent agreement). Nine of 10 borderline cases were positive when tested in triplicate. Conclusions The ddPCR of SARS-CoV-2 is an accurate method, with superior sensitivity for viral RNA detection. It could provide definitive evaluation of borderline positive cases or suspected false-negative cases.

scholarly journals Development of Quantitative Real-Time Polymerase Chain Reaction for Detection of and Discrimination between Erysipelothrix Rhusiopathiae and Other Erysipelothrix Species

2009 ◽  
Vol 21 (5) ◽  
pp. 701-706 ◽  
Author(s):  
Ho To ◽  
Tomohiro Koyama ◽  
Shinya Nagai ◽  
Kotaro Tuchiya ◽  
Tetsuo Nunoya
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Enrichment Culture ◽  
Bacterial Species ◽  
Erysipelothrix Rhusiopathiae ◽  
Chain Reaction ◽  
Tissue Samples ◽  
Practical Applications ◽  
Polymerase Chain

Quantitative real-time polymerase chain reaction (qPCR) assays were developed and validated in combination with enrichment culture for the detection and discrimination of Erysipelothrix rhusiopathiae and other Erysipelothrix species from tissue samples. The targets for SYBR green qPCR assays were the 16S ribosomal RNA gene for Erysipelothrix species and a gene involved in capsular formation for E. rhusiopathiae. The specificity of the assays was assessed with Erysipelothrix species and other related bacterial species. The limit of detection was found to be 5 colony-forming units per reaction. Amplification of DNA extracted from spleen and joint samples spiked with increasing quantities of Erysipelothrix cells was shown to be equally sensitive to DNA extracted from a pure bacterial culture. The assays were evaluated with 88 tissue samples from 3 experimentally infected pigs and 50 mice and with 36 tissue samples from 3 naturally infected pigs and 11 noninfected pigs. Results were compared with those of direct qPCR and conventional culture. The qPCR after enrichment increased the diagnostic sensitivity over that of culture and qPCR, thereby significantly reducing the total time taken for the detection of E. rhusiopathiae and other Erysipelothrix species. Therefore, this technique could be used for practical applications.

Comparison of Droplet Digital Polymerase Chain Reaction (ddPCR) and Real-Time Quantitative Polymerase Chain Reaction (qPCR) in Detecting Neonatal Invasive Fungal Infections

2021 ◽  
Vol 11 (3) ◽  
pp. 373-379
Author(s):  
Huitao Li ◽  
Xueyu Chen ◽  
Xiaomei Qiu ◽  
Weimin Huang ◽  
Chuanzhong Yang
Keyword(s):  
Polymerase Chain Reaction ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Fungal Isolation ◽  
Blood Samples ◽  
Fungal Strains ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Invasive fungal infection (IFI) is the leading cause of death in neonatal patients, yet the diagnosis of IFI remains a major challenge. At present, most IFI laboratory diagnostic methods are based on classical, but limited, methods such as fungal isolation and culture and histopathological examination. Recently, quantitative polymerase chain reaction (qPCR) and droplet digital polymerase chain reaction (ddPCR) technology have been adopted to quantify nucleic-acid identification. In this study, we established qPCR and ddPCR assays for IFI diagnosis and quantification. qPCR and ddPCR were carried out using identical primers and probe for the amplification of 18S rRNA. Assay results for three fungal strains were positive, whereas ten non-fungal strains had negative results, indicating 100% specificity for both ddPCR and qPCR methods. Genomic DNA of Candida albicans was tested after a serial dilution to compare the sensitivity of the two PCR methods. The limit of detection of ddPCR was 3.2 copies/L, which was a ten-fold increase compared with that of the qPCR method (32 copies/L). Blood samples from 127 patients with high-risk factors and clinical symptoms for IFI were collected from a NICU in Shenzhen, China, and analyzed using qPCR and ddPCR. Thirty-four blood samples from neonates had a proven or probable diagnosis of IFI, and 25 of these were positive by qPCR, whereas 30 were positive by ddPCR. Among the 93 blood samples from neonates who had a possible IFI or no IFI, 24 were positive using qPCR, and 7 were positive using ddPCR. In conclusion, ddPCR is a rapid and accurate pan-fungal detection method and provides a promising prospect for IFI clinical screening.

Quantitative detection of human enteric adenoviruses in river water by microfluidic digital polymerase chain reaction

2014 ◽  
Vol 70 (3) ◽  
pp. 555-560 ◽  
Author(s):  
Naohiro Kishida ◽  
Naohiro Noda ◽  
Eiji Haramoto ◽  
Mamoru Kawaharasaki ◽  
Michihiro Akiba ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
River Water ◽  
Water Samples ◽  
Real Time Pcr ◽  
Quantitative Detection ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction ◽  
Enteric Adenoviruses

We describe an assay for simple and accurate quantification of human enteric adenoviruses (EAdVs) in water samples using a recently developed quantification method named microfluidic digital polymerase chain reaction (dPCR). The assay is based on automatic distribution of reaction mixture into a large number of nanolitre-volume reaction chambers and absolute copy number quantification from the number of chambers containing amplification products on the basis of Poisson statistics. This assay allows absolute quantification of target genes without the use of standard DNA. Concentrations of EAdVs in Japanese river water samples were successfully quantified by the developed dPCR assay. The EAdVs were detected in seven of the 10 samples (1 L each), and the concentration ranged from 420 to 2,700 copies/L. The quantified values closely resemble those by most probable number (MPN)-PCR and real-time PCR when standard DNA was validated by dPCR whereas they varied substantially when the standard was not validated. Accuracy and sensitivity of the dPCR was higher than those of real-time PCR and MPN-PCR. To our knowledge, this is the first study that has successfully quantified enteric viruses in river water using dPCR. This method will contribute to better understanding of existence of viruses in water.

Detection of Naegleria fowleri, Acanthamoeba spp, and Balamuthia mandrillaris in Formalin-Fixed, Paraffin-Embedded Tissues by Real-Time Multiplex Polymerase Chain Reaction

2019 ◽  
Vol 152 (6) ◽  
pp. 799-807 ◽  
Author(s):  
Andrew P Norgan ◽  
Lynne M Sloan ◽  
Bobbi S Pritt
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Polymerase Chain ◽  
Formalin Fixed

Abstract Objectives Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. Methods FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. Results FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. Conclusions While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.

scholarly journals Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Unoh Ki ◽  
Takeru Suzuki ◽  
Satoshi Nakazawa ◽  
Yuuki Yonekawa ◽  
Kazuki Watanabe ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Confidence Interval ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Limit Of Detection ◽  
Quantitative Polymerase Chain Reaction ◽  
Chain Reaction ◽  
Low Copy Number ◽  
Polymerase Chain

AbstractRecently, in food safety and various other fields, qualitative and quantitative gene analysis using real-time polymerase chain reaction (PCR) method has become increasingly popular. The limit of detection (LOD) and quantifiable range for these measurements depends on the range and precision of DNA calibrators’ concentrations. Low-copy-number nucleic acid reference materials with low uncertainty produced by an inkjet system have been developed to allow for precise measurements in a low-copy-number region. However, when using a calibrator with a low copy number near one, the copy number distribution is asymmetric. Consequently, the confidence intervals of estimated copy numbers can include negative values when conventional methods of uncertainty estimation are used. A negative confidence interval is irrelevant in the context of copy number, which is always positive value or zero. Here, we propose a method to evaluate the uncertainty of real-time PCR measurements with representative values and an asymmetric 95% confidence interval. Moreover, we use the proposed method for the actual calculation of uncertainty of real-time PCR measurement results for low-copy-number DNA samples and demonstrate that the proposed method can evaluate the precision of real-time PCR measurements more appropriately in a low-copy-number region.

scholarly journals Considerations in Real-time Reverse Transcription Polymerase Chain Reaction (rRT-PCR) for the Detection of SARS-CoV-2 from Nasopharyngeal Swabs

2021 ◽  
pp. 68-78
Author(s):  
Priyadharshini Sekar ◽  
Godfred Antony Menezes ◽  
Pooja Shivappa ◽  
Biji Thomas George ◽  
Ashfaque Hossain
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Limit Of Detection ◽  
Disease Diagnosis ◽  
Antigen Detection ◽  
Disease Spread ◽  
Chain Reaction ◽  
The World ◽  
Polymerase Chain

Coronavirus Disease 2019 (COVID-19) was first reported in December 2019, in the City of Wuhan, China. Within the span of a few weeks, the disease had spread to other regions of China and eventually to different parts of the world. COVID 19 has affected 221 countries and territories around the world, with a total of 121,290,697 positive cases and 2,682,554 deaths as on March 17, 2021. Accurate disease diagnosis (for the SARS-Cov-2 virus and variants), coupled to patient isolation are currently critical strategies in restricting disease spread. Due to lack of time during this pandemic the diagnostics assays were not adequately validated. Infected individuals at times could potentially be missed by real-time reverse transcription polymerase chain reaction (rRT-PCR) for SARS-CoV-2 tests due to incorrect/inefficient sampling procedure, low limit of detection and epidemiology of the virus. rRT-PCR test results should be interpreted in conjunction with clinical examination and Computed Tomography (CT), particularly in suspected symptomatic individuals or those with epidemiological history of contact with known COVID-19 cases. Considering the above-mentioned constraints, the current scenario demands rapid and point-of-care tests for detection of SARS-CoV-2 in remote locations. To date, there is no reliable commercially available antigen detection kit. The infected subjects reveal low levels of antibodies against SARS-CoV-2 through the early period of infection. In addition, techniques such as, Digital RT-PCR technology and isothermal RNA amplification with electrochemical biosensors are some of the new technologies currently being developed to provide sensitive and specific SARS-Cov-2 antigen detection. The newly reported variant, SARS-CoV-2 VUI 202012/01 may not influence diagnostic outcomes as worldwide most PCR assays use two or more (including RdRp/ E/ N) reliable gene targets, besides S gene.

scholarly journals Establishment of Micro Droplet Digital Polymerase Chain Reaction and Real-Time Fluorescence Quantitative Polymerase Chain Reaction Technologies for Detecting Zika Virus

2018 ◽  
Author(s):  
Yuan Hui ◽  
Zhiming Wu ◽  
Zhiran Qin ◽  
Li Zhu ◽  
Junhe Liang ◽  
...  
Keyword(s):  
Cell Culture ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Zika Virus ◽  
Critical Role ◽  
Diagnostic Methods ◽  
Amplification Efficiency ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Digital Polymerase Chain Reaction

Establishment of diagnostic methods with low detection limits plays a critical role in the maintenance of early diagnosis, prevention of serious neurological complications, and control of the spread of ZIKA. In this study, we established the micro-droplet digital polymerase chain reaction (ddPCR) and real-time fluorescent quantification PCR (qPCR) protocols for the detection of Zika virus based on the NS5 gene. For the Zika standard plasmid, the standard curve of R2 was 0.999, and the amplification efficiency was 92.203%, as determined by qPCR. Both ddPCR and qPCR were positive for cell culture of Zika nucleic acid.The minimum detection limit of ddPCR is 1–2 times lower than qPCR. Moreover, all tests of Dengue virus (1–4 serotypes) were negative in cell culture. Overall, these results suggested than ddPCR may have a lower limit of detection than qPCR.

scholarly journals Quantitative Multiplex Real-Time Reverse Transcriptase–Polymerase Chain Reaction with Fluorescent Probe Detection of Killer Immunoglobulin-Like Receptors, KIR2DL4/3DL3

Diagnostics ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 588
Author(s):  
Wipaporn Wongfieng ◽  
Rungtiwa Nutalai ◽  
Amonrat Jumnainsong ◽  
Chanvit Leelayuwat
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Expression Patterns ◽  
Multiplex Assay ◽  
Clinical Samples ◽  
Lower Sensitivity ◽  
Chain Reaction ◽  
Kir Genes ◽  
Polymerase Chain

(1) Background: KIR2DL4/KIR3DL3 are the framework genes present in all KIR haplotypes, with unique expression patterns being present only in women and CD56bright NK cells. KIR genes have a high degree of DNA sequence identity. Consequently, they are one of the most challenging genes for molecular detection—especially regarding expressions; (2) Methods: We developed an effective method to determine KIR3DL3/KIR2DL4 expressions based on a multiplex quantitative real-time Reverse transcription polymerase chain reaction (qRT-PCR )with fluorescent probes using NK92; (3) Results: Standardizations of the singleplex KIR2DL4 and KIR3DL3 were performed to evaluate the sensitivity and specificity for further development of the multiplex assay. The limit of detection was at 500 copies each. There was cross-amplification with the presence of related KIR genes at a level of 5 × 107 copies. This is not biologically significant because this high level of KIR expression has not been found in clinical samples. The multiplex assay was reproducible equivalent to its singleplex (KIR2DL4; R2 = 0.995, KIR3DL3; R2 = 0.996, but lower sensitivity of 103 copies). Furthermore, the validation of the developed method on samples of blood donors showed high sensitivity (100%) and specificity (99.9%); (4) Conclusions: The developed method is reliable and highly specific suitable for evaluation of the KIR2DL4/3DL3 mRNA expressions in further applications.

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