Development of SYBR Green I-based real-time PCR assay for detection of drug resistance mutations in cytomegalovirus

ABSTRACT A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 102 CFU/cm2.

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Development of a SYBR Green I based one-step real-time PCR assay for the detection of Hantaan virus

Journal of Virological Methods ◽

10.1016/j.jviromet.2013.11.004 ◽

2014 ◽

Vol 196 ◽

pp. 145-151 ◽

Cited By ~ 12

Author(s):

Wei Jiang ◽

Ping-zhong Wang ◽

Hai-tao Yu ◽

Ye Zhang ◽

Ke Zhao ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Sybr Green ◽

Sybr Green I ◽

Hantaan Virus ◽

Pcr Assay ◽

One Step

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Fast and sensitive real-time PCR detection of major antiviral drug-resistance mutations in chronic hepatitis B patients using a predesigned panel of Locked-Nucleic-Acid TaqMan probes

Journal of Clinical Microbiology ◽

10.1128/jcm.00936-21 ◽

2021 ◽

Author(s):

Son V. Chu ◽

Son T. Vu ◽

Hang M. Nguyen ◽

Ngan T. Le ◽

Phuong T. Truong ◽

...

Keyword(s):

Drug Resistance ◽

Chronic Hepatitis ◽

Nucleic Acid ◽

Hepatitis B ◽

Real Time ◽

Chronic Hepatitis B ◽

Real Time Pcr ◽

Locked Nucleic Acid ◽

Resistance Mutations ◽

Drug Resistance Mutations

We developed a novel real-time PCR assay that simultaneously evaluates eleven major nucleos(t)ide antiviral (NA) drug-resistance mutations (mt) in chronic hepatitis B patients (CHB), including L180M, M204I/V, and V207M (lamivudine (LMV) resistance), N/H238A/T (Adefovir (ADF) resistance) which are circulating in Vietnam; T184G/L, S202I, M250V (entecavir (ETV) resistance), and A194T (tenofovir resistance) which have been recently reported in several studies across the globe. We detected drug-resistant mt in HBV samples using our predesigned panel of allele-specific locked-nucleic acid (LNA) probes. Our assay had a high sensitivity of 5% in a low HBV DNA population of ≥5 ×10 3 IU/mL and was validated in a cohort of 130 treatment-naive children and 98 NA-experienced adults with CHB. Single-point mt for LMV- and ADF-resistance were detected in 57.7% and 54.1% of the child and adult samples, respectively, with rtV207M (42.3%, children; 36.7%, adults) and rtN238T/A (15.4%, children; 16.3, adults) being the most frequent mt in these populations. Multiple-point mt including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I were only identified in two children, resulting in LMV-ADF-resistance and reduced ETV susceptibility. In conclusion, this assay accurately identified the mt profile of children (98.4%) and adults (91.2%) with CHB, which is comparable to established methods. This fast and sensitive screening method can be used for the detection of major NA-resistant mt circulating in developing countries, as well as providing a model for the development of similar mt-detection assays, especially for use in non-hospitalised patients who need their results within half a day before starting treatment.

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Development of a real-time PCR assay (SYBR Green I) for rapid identification and quantification of scyphomedusae Aurelia sp.1 planulae

Chinese Journal of Oceanology and Limnology ◽

10.1007/s00343-015-4091-0 ◽

2015 ◽

Vol 33 (4) ◽

pp. 974-987

Author(s):

Jianyan Wang ◽

Yu Zhen ◽

Tiezhu Mi ◽

Zhigang Yu ◽

Guoshan Wang

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Rapid Identification ◽

Sybr Green ◽

Sybr Green I ◽

Pcr Assay ◽

Identification And Quantification

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Real-time PCR assay for sensitive organ detection and epidemic investigation of Turbot reddish body iridovirus

Chinese Journal of Agricultural Biotechnology ◽

10.1017/s1479236209002605 ◽

2009 ◽

Vol 6 (1) ◽

pp. 61-67 ◽

Cited By ~ 2

Author(s):

Wu Cheng-Long ◽

Shi Cheng-Yin ◽

Huang Jie ◽

Kong Xiao-Yu

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Viral Genome ◽

Sybr Green ◽

Sybr Green I ◽

Quantitative Detection ◽

Rt Pcr ◽

Pcr Assay ◽

Linear Coefficient ◽

Sensitive Organ

AbstractA rapid and sensitive real-time polymerase chain reaction (PCR) assay coupled with SYBR Green I chemistry was developed for the quantitative detection of Turbot reddish body iridovirus (TRBIV) isolated from farmed turbot (Scophthalmus maximus). A 152 bp DNA fragment from the TRBIV major capsid protein (MCP) gene was involved in the real-time PCR (RT-PCR) assay using the Roter Gene 3000 sequence detection system. The PCR mixture contained a fluorescent dye, SYBR Green I, which exhibited fluorescence enhancement when bound to double-stranded (ds) DNA. The enhancement of fluorescence was proportional to the initial concentration of the template DNA. The positive control plasmid, pUCm-T/TRBIV MCP, containing the target sequence, was quantified to make a standard curve for sample detection after serial tenfold dilution. Linear coefficient correlations between the cycle threshold (CT) value and logarithmic positive plasmid concentration were close to one (R2=0.9952) and the detection limit of the assay was 102 copies of positive plasmids. The quantitative detection of virus in different tissues from TRBIV-infected fish showed that the spleen and kidney contained the largest number of viral particles (5.23×106 and 2.18×106 viral genome copies/mg tissue, respectively), while no viral DNA was detected in the muscular tissue. The molecular epidemic investigation of TRBIV showed that many cultured turbots were infected and TRBIV has become epidemic in turbot farms located along the Shandong peninsula. The virus number varied from 1.27×102 to 2.33×106 viral genome copies/mg tissue in spleens of infected turbot. These results suggest that the RT-PCR assay reported here can be used as a rapid, sensitive and quantitative method for TRBIV.

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