digital polymerase chain reaction
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2022 ◽  
Vol 12 ◽  
Author(s):  
Sergio Murolo ◽  
Marwa Moumni ◽  
Valeria Mancini ◽  
Mohamed Bechir Allagui ◽  
Lucia Landi ◽  
...  

Stagonosporopsis cucurbitacearum is an important seedborne pathogen of squash (Cucurbita maxima). The aim of our work was to develop a rapid and sensitive diagnostic tool for detection and quantification of S. cucurbitacearum in squash seed samples, to be compared with blotter analysis, that is the current official seed test. In blotter analysis, 29 of 31 seed samples were identified as infected, with contamination from 1.5 to 65.4%. A new set of primers (DB1F/R) was validated in silico and in conventional, quantitative real-time PCR (qPCR) and droplet digital (dd) PCR. The limit of detection of S. cucurbitacearum DNA for conventional PCR was ∼1.82 × 10–2 ng, with 17 of 19 seed samples positive. The limit of detection for ddPCR was 3.6 × 10–3 ng, which corresponded to 0.2 copies/μl. Detection carried out with artificial samples revealed no interference in the absolute quantification when the seed samples were diluted to 20 ng. All seed samples that showed S. cucurbitacearum contamination in the blotter analysis were highly correlated with the absolute quantification of S. cucurbitacearum DNA (copies/μl) in ddPCR (R2 = 0.986; p ≤ 0.01). Our ddPCR protocol provided rapid detection and absolute quantification of S. cucurbitacearum, offering a useful support to the standard procedure.


Plant Disease ◽  
2022 ◽  
Author(s):  
Roy Davis ◽  
Thomas Isakeit ◽  
Thomas Chappell

Fusarium wilt of cotton, caused by the soilborne fungal pathogen Fusarium oxysporum f. sp. vasinfectum (FOV), occurs in regions of the United States where cotton (Gossypium spp.) is grown. Race 4 of this pathogen (FOV4) is especially aggressive and does not require the co-occurrence of the root knot nematode (Meloidogyne incognita) to infect cotton. Its sudden appearance in far-west Texas in 2016 after many years of being restricted to California is of great concern, as is the threat of its continued spread through the cotton-producing regions of the United States. The aim of this research was to analyze the spatial variability of FOV4 inoculum density in the location where FOV4 is locally emerging, using quantitative and droplet digital polymerase chain reaction (qPCR and ddPCR) methods. Soil samples collected from a field with known FOV4 incidence in Fabens, Texas were analyzed. Appreciable variation in inoculum density was found to occur at spatial scales smaller than the size of plots involved in cultivar trial research, and was spatially autocorrelated (Moran’s I, Z = 17.73, p < 0.0001). These findings indicate that for cultivar trials, accounting for the spatial distribution of inoculum either by directly quantifying it or through the use of densely-distributed “calibration checks” is important to the interpretation of results.


Talanta ◽  
2022 ◽  
Vol 237 ◽  
pp. 122977
Author(s):  
Liping Xia ◽  
Jianjian Zhuang ◽  
Zheyu Zou ◽  
Juxin Yin ◽  
Ying Mu

Cancers ◽  
2021 ◽  
Vol 14 (1) ◽  
pp. 189
Author(s):  
Dora Raos ◽  
Irena Abramović ◽  
Miroslav Tomić ◽  
Alen Vrtarić ◽  
Tomislav Kuliš ◽  
...  

Seminoma (SE) is the most frequent type of testicular tumour, affecting predominantly young men. Early detection and diagnosis of SE could significantly improve life quality and reproductive health after diagnosis and treatment. Copy number variation (CNV) has already been associated with various cancers as well as SE. In this study, we selected four genes (MAGEC2, NANOG, RASSF1A, and KITLG) for CNV analysis in genomic DNA (gDNA), which are located on chromosomes susceptible to gains, and whose aberrant expression was already detected in SE. Furthermore, CNV was analysed in cell-free DNA (cfDNA) from seminal plasma. Analysis was performed by droplet digital polymerase chain reaction (ddPCR) on gDNA from SE and nonmalignant testicular tissue. Seminal plasma cfDNA from SE patients before and after surgery was analysed, as well as from healthy volunteers. The CNV hotspot in gDNA from SE tissue was detected for the first time in all analysed genes, and for two genes, NANOG and KITLG it was reflected in cfDNA from seminal plasma. Although clinical value is yet to be determined, presented data emphasize a potential use of CNV as a potential SE biomarker from a liquid biopsy.


2021 ◽  
Vol 8 ◽  
Author(s):  
Jie Yi ◽  
Nan Wang ◽  
Jie Wu ◽  
Yueming Tang ◽  
Jingjia Zhang ◽  
...  

Background:Pneumocystis jirovecii is a human-specific opportunistic fungus that causes Pneumocystis pneumonia (PCP), a life-threatening opportunistic lung infection that affects immunocompromised patients. P. jirovecii colonization may be linked to the transmission of the infection. The detection of P. jirovecii in immunocompromised patients is thus especially important. The low fungal load and the presence of PCR inhibitors limit the usefulness of quantitative PCR (qPCR) for accurate absolute quantification of P. jirovecii in specimens. Droplet digital PCR (ddPCR), however, presents a methodology that allows higher sensitivity and accuracy. Here, we developed a ddPCR method for detecting P. jirovecii DNA in respiratory specimens, and evaluated its sensitivity against qPCR.Materials and Methods: One bronchoalveolar fluid (BALF) sample each was collected from 82 patients with potential PCP to test the presence of P. jirovecii DNA using both ddPCR and qPCR, and samples with inconsistent results between the two methods were further tested by metagenomic next generation sequencing (mNGS). In addition, 37 sputum samples from 16 patients diagnosed with PCP, as well as continuous respiratory tract specimens from nine patients with PCP and treated with sulfonamides, were also collected for P. jirovecii DNA testing using both ddPCR and qPCR.Results: ddPCR and qPCR gave the same results for 95.12% (78/82) of the BALF samples. The remaining four specimens tested positive using ddPCR but negative using qPCR, and they were found to be positive by mNGS. Detection results of 78.37% (29/37) sputum samples were consistent between ddPCR and qPCR, while the other eight samples tested positive using ddPCR but negative using qPCR. The P. jirovecii load of patients with PCP decreased to undetectable levels after treatment according to qPCR, but P. jirovecii was still detectable using ddPCR.Conclusions: ddPCR was more sensitive than qPCR, especially at detecting low-pathogen-load P. jirovecii. Thus, ddPCR represents a useful, viable, and reliable alternative to qPCR in P. jirovecii testing in patients with immunodeficiency.


Micromachines ◽  
2021 ◽  
Vol 12 (12) ◽  
pp. 1562
Author(s):  
Xuee Chen ◽  
Qi Song ◽  
Beini Zhang ◽  
Yibo Gao ◽  
Kai Lou ◽  
...  

We designed a silicon-based fast-generated static droplets array (SDA) chip and developed a rapid digital polymerase chain reaction (dPCR) detection platform that is easy to load samples for fluorescence monitoring. By using the direct scraping method for sample loading, a droplet array of 2704 microwells with each volume of about 0.785 nL can be easily realized. It was determined that the sample loading time was less than 10 s with very simple and efficient characteristics. In this platform, a pressurized thermal cycling device was first used to solve the evaporation problem usually encountered for dPCR experiments, which is critical to ensuring the successful amplification of templates at the nanoliter scale. We used a gradient dilution of the hepatitis B virus (HBV) plasmid as the target DNA for a dPCR reaction to test the feasibility of the dPCR chip. Our experimental results demonstrated that the dPCR chip could be used to quantitatively detect DNA molecules. Furthermore, the platform can measure the fluorescence intensity in real-time. To test the accuracy of the digital PCR system, we chose three-channel silicon-based chips to operate real-time fluorescent PCR experiments on this platform.


10.2196/33746 ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. e33746
Author(s):  
Samir Badran ◽  
Ming Chen ◽  
John E Coia

Background Blood cultures are the cornerstone of diagnosis for detecting the presence of bacteria or fungi in the blood, with an average detection time of 48 hours and failure to detect a pathogen occurring in approximately 50% of patients with sepsis. Rapid diagnosis would facilitate earlier treatment and/or an earlier switch to narrow-spectrum antibiotics. Objective The aim of this study is to develop and implement a multiplex droplet digital polymerase chain reaction (ddPCR) assay as a routine diagnostic tool in the detection and identification of pathogens from whole blood and/or blood culture after 3 hours of incubation. Methods The study consists of three phases: (1) design of primer-probe pairs for accurate and reliable quantification of the most common sepsis-causing microorganisms using a multiplex reaction, (2) determination of the analytical sensitivity and specificity of the multiplex ddPCR assay, and (3) a clinical study in patients with sepsis using the assay. The QX200 Droplet Digital PCR System will be used for the detection of the following species-specific genes in blood from patients with sepsis: coa (staphylocoagulase) in Staphylococcus aureus, cpsA (capsular polysaccharide) in Streptococcus pneumoniae, uidA (beta-D-glucuronidase) in Escherichia coli, oprL (peptidoglycan-associated lipoprotein) in Pseudomonas aeruginosa, and the highly conserved regions of the 16S rRNA gene for Gram-positive and Gram-negative bacteria. All data will be analyzed using QuantaSoft Analysis Pro Software. Results In phase 1, to determine the optimal annealing temperature for the designed primer-probe pairs, results from a gradient temperature experiment will be collected and the limit of detection (LOD) of the assay will be determined. In phase 2, results for the analytical sensitivity and specificity of the assay will be obtained after an optimization of the extraction and purification method in spiked blood. In phase 3, clinical sensitivity and specificity as compared to the standard blood culture technique will be determined using 301 clinical samples. Conclusions Successful design of primer-probe pairs in the first phase and subsequent optimization and determination of the LOD will allow progression to phase 3 to compare the novel method with existing blood culture methods. International Registered Report Identifier (IRRID) PRR1-10.2196/33746


Hematology ◽  
2021 ◽  
Vol 2021 (1) ◽  
pp. 320-330
Author(s):  
Ilaria Del Giudice ◽  
Irene Della Starza ◽  
Robin Foà

Abstract Among indolent non-Hodgkin lymphomas (iNHLs), the analysis of measurable/minimal residual disease (MRD) has been extensively applied to follicular lymphoma (FL). Treatment combinations have deeply changed over the years, as well as the techniques to measure MRD, which is currently evaluated only in the setting of clinical trials. Here, we discuss the evidence on the role of molecular monitoring in the management of FL. Mature data support the quantification of molecular tumor burden at diagnosis as a tool to stratify patients in risk categories and of MRD evaluation at the end of treatment to predict progression-free survival and overall survival. Moreover, MRD deserves further studies as a tool to refine the clinical/metabolic response and to modulate treatment intensity/duration. Patients with a higher relapse probability can be identified, but the relevance of continuous molecular follow-up should be clarified by kinetic models of MRD analysis. Being the BCL2/heavy chain immunoglobulin gene hybrid rearrangement detectable in about 50% to 60% of advanced FL and in 30% of positron emission tomography/computed tomography–staged localized FL, technical advancements such as next-generation sequencing/target locus amplification may allow broadening the FL population carrying a molecular marker. Droplet digital polymerase chain reaction can better quantify MRD at low levels, and novel sources of DNA, such as cell-free DNA, may represent a noninvasive tool to monitor MRD. Finally, MRD in other iNHLs, such as lymphoplasmacytic lymphoma/Waldenström macroglobulinemia and marginal zone lymphoma, is beginning to be explored.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zhi Yao Ma ◽  
Cherry Sze Yan Chan ◽  
Kam Shing Lau ◽  
Lui Ng ◽  
Yuen Yee Cheng ◽  
...  

AbstractMethylated septin 9 (SEPT9) has been approved for non-invasive screening of colorectal cancer (CRC), but data on monitoring of CRC is sparse. Droplet digital polymerase chain reaction (ddPCR), with higher detection precision and simpler quantification than conventional PCR, has not been applied in SEPT9 detection. We explored the role of SEPT9 ddPCR for CRC detection and to measure serial SEPT9 levels in blood samples of CRC patients before and 3-month after surgery. SEPT9 methylated ratio, methylated abundance, and CEA levels were all higher in CRC patients than normal controls (all P < 0.05). The area under the curve (AUC) for methylated ratio and abundance to detect CRC was 0.707 and 0.710, respectively. There was an increasing trend for SEPT9 methylated abundance from proximal to distal cancers (P = 0.017). At 3-month after surgery, both methylated abundance and ratio decreased (P = 0.005 and 0.053, respectively), especially methylated abundance in stage III and distal cancer (both P < 0.01). We have developed a ddPCR platform for the quantitative detection of plasma SEPT9 in CRC patients. SEPT9 methylated abundance had an early post-operative decline, which may be useful in monitoring of treatment response.


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