quantitative reverse transcription pcr
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2021 ◽  
Vol 12 ◽  
Author(s):  
Feiyang Zhang ◽  
Qin Li ◽  
Jiawei Bai ◽  
Manlin Ding ◽  
Xiangjin Yan ◽  
...  

Heteroresistance can lead to treatment failure and is difficult to detect by the methods currently employed by clinical laboratories. The aim of this study was to investigate the prevalence of the amikacin-heteroresistant Klebsiella pneumoniae strains and explore potential amikacin heteroresistance mechanism through whole-genome sequencing (WGS) and quantitative reverse-transcription PCR (qRT-PCR). In this study, 13 isolates (8.39%) were considered as amikacin-heteroresistant K. pneumoniae strains among a total of 155 K. pneumoniae strains. The majority of the heterogeneous phenotypes (11/13, 84.61%) was unstable and the minimal inhibitory concentrations (MICs) fully or partially reverted back to the level of susceptibility of the parental isolate. The frequency of heteroresistant subpopulation ranged from 2.94×10−7 to 5.59×10−6. Whole-genome sequencing and single-nucleotide variants (SNVs) analysis showed that there were different nucleotide and resultant amino acid alterations among an amikacin-heteroresistant strain S38 and the resistant subpopulation S38L in several genes. Quantitative reverse-transcription PCR analysis revealed that the increased expression of aminoglycoside resistance genes detected in amikacin-heteroresistant K. pneumoniae strains might be associated with amikacin heteroresistance. The findings raise concerns for the emergence of amikacin-heteroresistant K. pneumoniae strains and the use of amikacin as therapy for the treatment of multidrug-resistant K. pneumoniae strains.


2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Florian Wagner ◽  
Ralf Greim ◽  
Kathrin Krebs ◽  
Finn Luebben ◽  
Arno Dimmler

Abstract Background Fusions of neurotrophic tropomyosin receptor kinase genes NTRK1, NTRK2 and NTRK3 with various partner genes occur in both common and rare tumours and are of paramount predictive value due to the availability of very effective pan-Trk inhibitors like Larotrectinib and Entrectinib. Detection of NTRK fusions is mainly performed by fluorescence in situ hybridization (FISH) and next generation sequencing (NGS). The case described here showed a very unusual, but highly significant FISH signal pattern with an NTRK3 break apart probe, indicative of a functional NTRK3 rearrangement. Case presentation We describe here the case of a male patient who was originally diagnosed with an adenocarcinoma of the parotid gland without evidence of metastases. After the development of multiple lung metastases, an extensive immunohistochemical and molecular examination of archived tumour tissue including analysis of NTRK was performed. NTRK expression was detected by immunohistochemistry (IHC) and then comprehensively analysed further by FISH, quantitative reverse transcription PCR (RT-qPCR), and NGS. NTRK3 break apart FISH showed multiple and very faint single 3′ signals in addition to fusion signals. Quantitative reverse transcription PCR and NGS confirmed an ETV6:exon5-NTRK3:exon15 fusion. Diagnosis was therefore revised to metastatic secretory carcinoma of the salivary gland, and the patient subsequently treated with Larotrectinib, resulting in persisting partial remission. Conclusions Our findings underline the importance to be aware of non-canonical signal patterns during FISH analysis for detection of NTRK rearrangements. Very faint single 3′ signals can indicate a functional NTRK rearrangement and therefore be of high predictive value.


Heliyon ◽  
2021 ◽  
pp. e07560
Author(s):  
SungJun Park ◽  
Cheonghoon Lee ◽  
Kyuseon Cho ◽  
Hye Young Ko ◽  
Sung Jae Jang ◽  
...  

2021 ◽  
Author(s):  
Chao Tan ◽  
Xi Zeng ◽  
Meile Mo ◽  
Xiaoyun Ma ◽  
Qiuli Liang ◽  
...  

Aim: To explore the expression profiles of long noncoding RNAs (lncRNAs) and identify novel lncRNAs as biomarkers for early diagnosis and therapy of hepatocellular carcinoma (HCC). Materials & methods: Expression profiles of lncRNAs and mRNAs in five paired HCC and adjacent normal tissues were obtained by RNA sequencing. Eight lncRNAs, including two novel liver-specific lncRNAs (NONHSAT059247.2 and NONHSAT013897.2), were validated in another 74 pairs of HCC and adjacent normal tissues by quantitative reverse transcription PCR. Results: The results of quantitative reverse transcription PCR showed that NONHSAT252133.1, NONHSAT112116.2 and NONHSAT242657.1 were significantly upregulated in HCC tissues, whereas NONHSAT169790.1, NONHSAT059247.2 and NONHSAT013897.2 were significantly downregulated. Two liver-specific lncRNAs demonstrated excellent diagnostic performance: NONHSAT059247.2 (area under the curve = 0.941, 95% CI: 0.902–0.979, p < 0.0001), NONHSAT013897.2 (area under the curve = 0.944, 95% CI: 0.906–0.983, p < 0.0001). Conclusion: The liver-specific lncRNAs NONHSAT059247.2 and NONHSAT013897.2, may provide new biomarkers for the future study on diagnosis, therapy, and mechanisms of HCC.


2020 ◽  
Author(s):  
Yi-Hui Gu ◽  
Xi-Wei Cui ◽  
Jie-Yi Ren ◽  
Man-Mei Long ◽  
Wei Wang ◽  
...  

AbstractTranscriptomics has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the most qualified reference genes for the relative quantitation analysis of transcriptomics and real-time quantitative reverse-transcription PCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes (RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC and RPL32) were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most used internal reference gene, was significantly unstable. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. Our recommended internal reference genes may improve the accuracy and reproducibility of the results of transcriptomics and real-time quantitative reverse-transcription PCR in further gene expression analyses of NF1 related tumors.


2020 ◽  
Author(s):  
Mai Niikura ◽  
Satomi Atobe ◽  
Akira Takahashi ◽  
Yukiko Kado ◽  
Takuya Sugimoto ◽  
...  

Abstract Background: For Pseudomonas aeruginosa, nosocomial infection control and appropriate antimicrobial treatment have become important issues. Diagnosis is critical in managing P. aeruginosa infection, but conventional methods are not highly accurate or rapid. A novel P. aeruginosa quantification system based on 23S rRNA-targeted quantitative reverse transcription PCR (qRT-PCR) is a candidate diagnostic tool for managing P. aeruginosa infection. Here, we assessed the performance and potential impact of qRT-PCR on antibiotic therapy administered to ICU patients.Methods: We first evaluated specificity and detection sensitivity of the 23S rRNA-targeted qRT-PCR system in vitro and determined whether P. aeruginosa viable counts detected by this system reflect the inflammatory response of infected cells. Next, we utilized this system on fecal samples collected from 65 septic ICU patients and 44 healthy volunteers to identify the ICU infection status. Additionally, we monitored drug-resistant P. aeruginosa in 4 ICU patients. The trend was compared with trends in fecal microbiota composition, antibiotic use, and mechanical ventilator use.Results: The 23S rRNA-targeted qRT-PCR system quantified P. aeruginosa directly from clinical samples with high sensitivity (blood, 1 cell/mL; stool, 100 cells/g) without cross-reaction (within 6 h). Additionally, P. aeruginosa numbers detected under antibacterial treatment correlated well with the inflammatory response compared to other detection methods such as culturing and qPCR. Using this system, we confirmed that the P. aeruginosa detection ratio in ICU patients was significantly higher than that in healthy volunteers (49.2% vs. 13.6%, P<0.05). While some ICU patients had high P. aeruginosa numbers in feces (108 cells/g of feces), some patients had very low P. aeruginosa numbers (102 cells/g of feces) as observed in healthy volunteers. P. aeruginosa counts in feces of ICU patients monitored by this system correlated negatively with the proportion of total obligate anaerobes and positively with facultative anaerobes and aerobes. Additionally, trends in P. aeruginosa counts accurately reflected various treatment backgrounds of ICU patients.Conclusion: Our results suggest that this new 23S rRNA-targeted qRT-PCR system is beneficial for early diagnosis and evaluation of appropriate antibacterial treatment and may be a useful tool in combating P. aeruginosa infection.


2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Mayu Nagura-Ikeda ◽  
Kazuo Imai ◽  
Sakiko Tabata ◽  
Kazuyasu Miyoshi ◽  
Nami Murahara ◽  
...  

ABSTRACT The clinical performances of six molecular diagnostic tests and a rapid antigen test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were clinically evaluated for the diagnosis of coronavirus disease 2019 (COVID-19) in self-collected saliva. Saliva samples from 103 patients with laboratory-confirmed COVID-19 (15 asymptomatic and 88 symptomatic) were collected on the day of hospital admission. SARS-CoV-2 RNA in saliva was detected using a quantitative reverse transcription-PCR (RT-qPCR) laboratory-developed test (LDT), a cobas SARS-CoV-2 high-throughput system, three direct RT-qPCR kits, and reverse transcription–loop-mediated isothermal amplification (RT-LAMP). The viral antigen was detected by a rapid antigen immunochromatographic assay. Of the 103 samples, viral RNA was detected in 50.5 to 81.6% of the specimens by molecular diagnostic tests, and an antigen was detected in 11.7% of the specimens by the rapid antigen test. Viral RNA was detected at significantly higher percentages (65.6 to 93.4%) in specimens collected within 9 days of symptom onset than in specimens collected after at least 10 days of symptoms (22.2 to 66.7%) and in specimens collected from asymptomatic patients (40.0 to 66.7%). Self-collected saliva is an alternative specimen option for diagnosing COVID-19. The RT-qPCR LDT, a cobas SARS-CoV-2 high-throughput system, direct RT-qPCR kits (except for one commercial kit), and RT-LAMP showed sufficient sensitivities in clinical use to be selectively used in clinical settings and facilities. The rapid antigen test alone is not recommended for an initial COVID-19 diagnosis because of its low sensitivity.


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