scholarly journals Method for Solving Non-specific Amplification Interference of Fluorescence Quantitative PCR in Gene Detection

2020 ◽  
Vol 4 (6) ◽  
Author(s):  
Jinku Zhang ◽  
Jirui Sun ◽  
Haizhi Qiao ◽  
Lu Han ◽  
Yunjia Liu
Keyword(s):  
Quantitative Pcr ◽  
Melting Curve ◽  
Fluorescence Signal ◽  
Detection Accuracy ◽  
Gene Detection ◽  
Step Method ◽  
Application Range ◽  
Fluorescence Quantitative Pcr ◽  
Specific Amplification ◽  
Improved Accuracy

Objective: To explore a method to solve the issue of interference in fluorescence quantitative PCR non-specific amplification for gene detection. Method: A three-step method was used for amplification, and the quantitative fluorescence signal collection process was set in the extension stage. Results: Three-step amplification has the advantages of wide application range; improved accuracy; and reduced primer design requirements. Conclusion: The interference of non-specific amplification signals was effectively avoided, the melting curve plotting process was omitted, the reaction time was shortened, and the detection accuracy was improved.

Establishment and Preliminary Application of Multiplex Fluorescent Quantitative PCR for Simultaneous Detection of BVDV, BRV and BCV

2021 ◽  
Author(s):  
Liyun Chang ◽  
Zhiyong Liu ◽  
Yuelan Zhao ◽  
Yan Li ◽  
Jianhua Qin
Keyword(s):  
Quantitative Pcr ◽  
Quantitative Methods ◽  
Diarrheal Disease ◽  
Quantitative Polymerase Chain Reaction ◽  
Simultaneous Detection ◽  
Cross Reactivity ◽  
N Gene ◽  
Established Method ◽  
Diarrhea Virus ◽  
Fluorescence Quantitative Pcr

Background: In this study, we aimed to establish a multiplex fluorescence quantitative polymerase chain reaction (PCR) method for the identification and detection of bovine viral diarrhea virus (BVDV), bovine rotavirus (BRV) and bovine coronavirus (BCV). Methods: Based on the highly conserved sequences of BVDV E2 gene, BRV VP6 gene and BCV N gene in GenBank, specific primers were designed to amplify the target gene fragments of each virus and the reaction conditions and system were optimized. Multiple fluorescence quantitative methods were established by fluorescence quantitative PCR. Result: The minimum detection limits of plasmid standards for BVDV, BRV and BCV by multiplex fluorescence quantitative PCR were 1.19×102 copies/μL, 3.89×101 copies/μL and 3.74×101 copies/μL, respectively. The lowest sensitivity of the established method was 100 times higher than that of conventional PCR and had high sensitivity. Furthermore, BVDV, BRV and BCV were amplified specifically, with no cross-reactivity with Escherichia coli (E. coli), Salmonella and infectious bovine rhinotracheitis virus (IBRV). The intra-and inter-group coefficients of variation were less than 1%, showing good assay repeatability. Using the established method and ordinary multiplex PCR to simultaneously detect 150 clinical diarrheal disease material samples, the coincidence rate of samples with mixed infection of the three viruses was 83.3%. The results showed that the multiplex fluorescent quantitative PCR detection method established in this study provides a rapid, sensitive and specific technique for clinical diagnosis and epidemiological monitoring of BVDV, BRV and BCV.

scholarly journals Identification of Burkholderia cepacia complex pathogens by rapid-cycle PCR with fluorescent hybridization probes

2006 ◽  
Vol 55 (6) ◽  
pp. 721-727 ◽  
Author(s):  
Ralf-Peter Vonberg ◽  
Susanne Häußler ◽  
Peter Vandamme ◽  
Ivo Steinmetz
Keyword(s):  
Burkholderia Cepacia ◽  
Melting Curve ◽  
Resonance Energy ◽  
Burkholderia Cenocepacia ◽  
Burkholderia Cepacia Complex ◽  
Melting Curve Analysis ◽  
Identification System ◽  
Fluorescence Signal ◽  
Pathogenic Potential ◽  
Specific Fluorescence

Members of the Burkholderia cepacia complex are important bacterial pathogens in cystic fibrosis (CF) patients. The B. cepacia complex currently consists of nine genetic subgroups (genomovars) of different epidemiological relevance and possibly of different pathogenic potential in humans. In this study, a new approach was developed for the rapid identification of B. cepacia genomovar I, Burkholderia multivorans (genomovar II), Burkholderia cenocepacia (lineage III-A and III-B), Burkholderia stabilis (genomovar IV) and Burkholderia vietnamiensis (genomovar V), which cause the large majority of infections in CF patients. The method was based on the detection of differences in the recA gene sequence by using rapid-cycle PCR and genomovar-specific fluorescence resonance energy transfer (FRET) probes. The genomovar status of all 39 B. cepacia complex strains tested (genomovars I–V) was identified by melting-curve analysis. Each FRET probe produced a specific fluorescence signal only with the respective genomovar, and not with other B. cepacia complex strains and Burkholderia spp. The identification system was easy to handle and revealed B. cepacia complex genomovar I–V status from culture isolates within about 1 h.

scholarly journals TWO-STEP PROVIDING OF DESIRED QUALITY IN LOSSY IMAGE COMPRESSION BY SPIHT

2020 ◽  
pp. 22-32
Author(s):  
Fangfang Li ◽  
Sergey Krivenko ◽  
Vladimir Lukin
Keyword(s):  
Image Compression ◽  
Visual Quality ◽  
Lossy Compression ◽  
Single Step ◽  
Set Partitioning ◽  
Discrete Wavelet ◽  
Compression Method ◽  
Step Method ◽  
Application Range ◽  
Special Cases

Image information technology has become an important perception technology considering the task of providing lossy image compression with the desired quality using certain encoders Recent researches have shown that the use of a two-step method can perform the compression in a very simple manner and with reduced compression time under the premise of providing a desired visual quality accuracy. However, different encoders have different compression algorithms. These issues involve providing the accuracy of the desired quality. This paper considers the application of the two-step method in an encoder based on a discrete wavelet transform (DWT). In the experiment, bits per pixel (BPP) is used as the control parameter to vary and predict the compressed image quality, and three visual quality evaluation metrics (PSNR, PSNR-HVS, PSNR-HVS-M) are analyzed. In special cases, the two-step method is allowed to be modified. This modification relates to the cases when images subject to lossy compression are either too simple or too complex and linear approximation of dependences is no more valid. Experimental data prove that, compared with the single-step method, after performing the two-step compression method, the mean square error of differences between desired and provided values drops by an order of magnitude. For PSNR-HVS-M, the error of the two-step method does not exceed 3.6 dB. The experiment has been conducted for Set Partitioning in Hierarchical Trees (SPIHT), a typical image encoder based on DWT, but it can be expected that the proposed method applies to other DWT-based image compression techniques. The results show that the application range of the two-step lossy compression method has been expanded. It is not only suitable for encoders based on discrete cosine transform (DCT) but also works well for DWT-based encoders.

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