Development and Efficacy of Droplet Digital PCR for Detection of Strongyloides stercoralis in Stool

2021 ◽  
Author(s):  
Kantapong Iamrod ◽  
Apisit Chaidee ◽  
Rucksak Rucksaken ◽  
Kulthida Y. Kopolrat ◽  
Chanika Worasith ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Neglected Tropical Diseases ◽  
Agar Plate ◽  
High Sensitivity ◽  
Strongyloides Stercoralis ◽  
Digital Pcr ◽  
Immunosuppressive Drug ◽  
Stool Examination ◽  
Concentration Technique

Human strongyloidiasis is one of the neglected tropical diseases caused by infection with soil-transmitted helminth Strongyloides stercoralis. Conventional stool examination, a method commonly used for diagnosis of S. stercoralis, has low sensitivity, especially in the case of light infections. Herein, we developed the droplet digital polymerase chain reaction (ddPCR) assay to detect S. stercoralis larvae in stool and compared its performance with real-time PCR and stool examination techniques (formalin ethyl-acetate concentration technique [FECT] and agar plate culture [APC]). The ddPCR results showed 98% sensitivity and 90% specificity, and real-time PCR showed 82% sensitivity and 76.7% specificity when compared with the microscopic methods. Moreover, ddPCR could detect a single S. stercoralis larva in feces, and cross-reactions with other parasites were not observed. In conclusion, a novel ddPCR method exhibited high sensitivity and specificity for detection of S. stercoralis in stool samples. This technique may help to improve diagnosis, particularly in cases with light infection. In addition, ddPCR technique might be useful for screening patients before starting immunosuppressive drug therapy, and follow-up after treatment of strongyloidiasis.

Using of Digital Droplet PCR in the detection of Mycobacterium tuberculosis DNA with FFPE and Cytological samples

2020 ◽  
Author(s):  
Ziyang Cao ◽  
Wei Wu ◽  
Haiting Wei ◽  
Caixia Gao ◽  
Liping Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Digital Pcr ◽  
Digital Droplet Pcr ◽  
Gray Area ◽  
Droplet Pcr ◽  
Spss Software ◽  
Tuberculosis Therapy ◽  
Formalin Fixed

Abstract Backgrounds: Accurate diagnosis for TB is essential for TB control. Droplet digital PCR (ddPCR) is a technology that has high sensitivity. However, the use of ddPCR for the detection of TB pathogen in pathological samples is not been fully studied.Methods: A total of 88 samples from the patients who were suspected of tuberculosis were involved in this study, including 65 formalin-fixed and paraffin-embedded (FFPE) specimens and 22 cytological samples. Digital Droplet PCR was used to compare the sensitivity with Real-time PCR. The real-time PCR ct value and ddPCR TB abundant ratio were analyzed by SPSS software.Results: Among the 62 samples that were “possible TB” detected by real-time PCR, 34 samples were ddPCR-positive, 18 samples were ddPCR-negative, and 10 samples were in “gray area” by ddPCR. 26 patients that were ddPCR-positive received anti-tuberculosis therapy and 14 cases of them benefit from the treatment.Conclusions: ddPCR is more sensitive in the detection of TB than Real-time PCR. ddPCR methods can be used as an additional means for the diagnosis of TB from pathological samples.

scholarly journals Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

2021 ◽  
Author(s):  
Christian Schulze ◽  
Anne-Catrin Geuthner ◽  
Dietrich Mäde
Keyword(s):  
Durum Wheat ◽  
Real Time ◽  
Bread Wheat ◽  
Common Wheat ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Food Fraud ◽  
Single Genome ◽  
Cereal Species

AbstractFood fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

scholarly journals Single-copy detection of somatic variants from solid and liquid biopsy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ana-Luisa Silva ◽  
Paulina Klaudyna Powalowska ◽  
Magdalena Stolarek ◽  
Eleanor Ruth Gray ◽  
Rebecca Natalie Palmer ◽  
...  
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Real Time Pcr ◽  
Clinical Decision Making ◽  
High Sensitivity ◽  
Clinical Decision ◽  
Single Copy ◽  
Turnaround Time ◽  
Copy Detection ◽  
Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

Detection of transgenic rice line TT51-1 in processed foods using conventional PCR, real-time PCR, and droplet digital PCR

Food Control ◽  
2019 ◽  
Vol 98 ◽  
pp. 380-388 ◽  
Author(s):  
Xiaofu Wang ◽  
Ting Tang ◽  
Qingmei Miao ◽  
Shilong Xie ◽  
Xiaoyun Chen ◽  
...  
Keyword(s):  
Real Time ◽  
Transgenic Rice ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Processed Foods ◽  
Conventional Pcr ◽  
Rice Line ◽  
Transgenic Rice Line

Exploring a phage-based real-time PCR assay for diagnosing Acinetobacter baumannii bloodstream infections with high sensitivity

2018 ◽  
Vol 1044 ◽  
pp. 147-153 ◽  
Author(s):  
Jun Luo ◽  
Mengwei Jiang ◽  
Jin Xiong ◽  
Junhua Li ◽  
Xiaoxu Zhang ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Real Time ◽  
Real Time Pcr ◽  
Bloodstream Infections ◽  
High Sensitivity ◽  
Pcr Assay

42P Method optimization for the detection of chimerism by real-time PCR and droplet digital PCR

2021 ◽  
Vol 32 ◽  
pp. S1358
Author(s):  
I.M. Lambrescu ◽  
V.S. Ionescu ◽  
G. Gaina ◽  
A. Popa ◽  
C. Niculite ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Method Optimization

Evaluation of droplet digital PCR for the detection of black canker disease in tomato

Plant Disease ◽  
2021 ◽  
Author(s):  
Li Wang ◽  
Tian Qian ◽  
Pei Zhou ◽  
Wenjun Zhao ◽  
Xianchao Sun
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Pathogens ◽  
Plant Protection ◽  
Detection Threshold ◽  
Digital Pcr ◽  
Taqman Probe ◽  
Tomato Seed ◽  
Canker Disease ◽  
Probe Set

Clavibacter michiganensis subsp. michiganensis (Cmm), the cause of bacterial canker disease, is one of the most destructive pathogens in greenhouse and field tomato. The pathogen is now present in all main production areas of tomato and is quite widely distributed in the EPPO（European and Mediterranean Plant Protection Organization）region. The inspection and quarantine of the plant pathogens relies heavily on accurate detection tools. Primers and probes reported in previous studies do not distinguish the Cmm pathogen from other closely related subspecies of C. michiganensis, especially the non-pathogenic subspecies that were identified from tomato seeds recently. Here, we have developed a droplet digital polymerase chain reaction (ddPCR) method for the identification of this specific bacterium with primers/TaqMan probe set designed based on the pat-1 gene of Cmm. This new primers/probe set has been evaluated by qPCRthe real time PCR（qPCR） and ddPCR. The detection results suggest that the ddPCR method established in this study was highly specific for the target strains. The result showed the positive amplification for all 5 Cmm strains，and no amplification was observed for the other 43 tested bacteria, including the closely related C. michiganensis strains. The detection threshold of ddPCR was 10.8 CFU/mL for both pure Cmm cell suspensions and infected tomato seed, which was 100 times-fold more sensitive than that of the real-time PCR (qPCR ) performed using the same primers and probe. The data obtained suggest that our established ddPCR could detect Cmm even with low bacteria load, which could facilitate both Cmm inspection for pathogen quarantine and the routine pathogen detection for disease control of black canker in tomato.

Reverse transcriptase droplet digital PCR vs reverse transcriptase quantitative real‐time PCR for serum HBV RNA quantification

2020 ◽  
Vol 92 (12) ◽  
pp. 3365-3372 ◽  
Author(s):  
Umaporn Limothai ◽  
Natthaya Chuaypen ◽  
Kittiyod Poovorawan ◽  
Watcharasak Chotiyaputta ◽  
Tawesak Tanwandee ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Real Time ◽  
Real Time Pcr ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Quantitative Real Time Pcr ◽  
Rna Quantification

scholarly journals Neutralization Assay for Zika and Dengue Viruses by Use of Real-Time-PCR-Based Endpoint Assessment

2017 ◽  
Vol 55 (10) ◽  
pp. 3104-3112 ◽  
Author(s):  
Heather L. Wilson ◽  
Thomas Tran ◽  
Julian Druce ◽  
Myrielle Dupont-Rouzeyrol ◽  
Michael Catton
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
High Sensitivity ◽  
Neutralization Assay ◽  
Cross Reactivity ◽  
Health Concern ◽  
Confirmatory Test ◽  
Virus Neutralization Assay ◽  
History Of ◽  
Infective Complications

ABSTRACTThe global spread and infective complications of Zika virus (ZKV) and dengue virus (DENV) have made them flaviviruses of public health concern. Serological diagnosis can be challenging due to antibody cross-reactivity, particularly in secondary flavivirus infections or when there is a history of flavivirus vaccination. The virus neutralization assay is considered to be the most specific assay for measurement of anti-flavivirus antibodies. This study describes an assay where the neutralization endpoint is measured by real-time PCR, providing results within 72 h. It demonstrated 100% sensitivity (24/24 ZKV and 15/15 DENV) and 100% specificity (11/11 specimens) when testing well-characterized sera. In addition, the assay was able to determine the correct DENV serotype in 91.7% of cases. The high sensitivity and specificity of the real-time PCR neutralization assay makes it suitable to use as a confirmatory test for sera that are reactive in commercial IgM/IgG enzyme immunoassays. Results are objective and the PCR-based measurement of the neutralization endpoint lends itself to automation so that throughput may be increased in times of high demand.

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