Direct digital polymerase chain reaction chip for the detection of EGFR T790M mutation in plasma

Talanta ◽  
2022 ◽  
Vol 237 ◽  
pp. 122977
Author(s):  
Liping Xia ◽  
Jianjian Zhuang ◽  
Zheyu Zou ◽  
Juxin Yin ◽  
Ying Mu
Author(s):  
Jing Xu ◽  
Timothy Kirtek ◽  
Yan Xu ◽  
Hui Zheng ◽  
Huiyu Yao ◽  
...  

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.


2021 ◽  
Vol 11 (3) ◽  
pp. 373-379
Author(s):  
Huitao Li ◽  
Xueyu Chen ◽  
Xiaomei Qiu ◽  
Weimin Huang ◽  
Chuanzhong Yang

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.


In Vivo ◽  
2019 ◽  
Vol 33 (6) ◽  
pp. 2273-2280 ◽  
Author(s):  
HYUNKYUNG PARK ◽  
DONG-YEOP SHIN ◽  
INHO KIM ◽  
SANG-KYUN SOHN ◽  
YOUNGIL KOH ◽  
...  

2020 ◽  
Vol 167 (10) ◽  
Author(s):  
Janine Wäge ◽  
Oliver Schmale ◽  
Matthias Labrenz

Abstract Methane enrichments are frequently observed in the oxic upper water column of the central Baltic Sea during summer months. However, methane sources as well as the fate of methane produced in surface near waters still remain unclear. In the present study, we conducted ship-based grazing experiments to examine the presence of methanogenic archaea in copepod faecal pellets. We quantified bacterial and archaeal 16S rRNA and the mcrA gene and transcripts within copepod faecal pellets by using droplet digital polymerase chain reaction. We showed that the pellets (< 150-µm) harbour a small number of methanogenic archaea; however, mcrA transcripts indicating methanogenic activity were not detected. This suggests that copepod faecal pellets from the central Baltic Sea, similar to analogous data on copepod guts, harbour the potential but are an unlikely hotspot for methane production by methanogenic archaea.


2020 ◽  
Vol 11 ◽  
Author(s):  
Carolina Villamil ◽  
Martha Nancy Calderon ◽  
Maria Mercedes Arias ◽  
John Emerson Leguizamon

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