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Multiplex quadruple bioluminescent assay system.

ABSTRACT: Bioluminescence (BL) is unique cold body radiation of light, generated by luciferin-luciferase reactions and commonly used in various bioassays and molecular imaging. However, most of the peak emissions of BL populate the blue-yellow region and have broad spectral bandwidths and thus superimpose each other, causing optical cross-leakages in multiplex assays. This study synthesized a new series of coelenterazine (CTZ) analogues, named K-series, that selectively illuminates marine luciferases with unique, blue-shifted spectral properties. The optical property and specificity of the K-series CTZ analogues were characterized by marine luciferases, with K2 and K5 found to specifically luminesce with ALuc- and RLuc-series marine luciferases, respectively. The results confirmed that the luciferase specificity and color variation of the CTZ analogues minimize the cross-leakages of BL signals and enable high-throughput screening of specific ligands in the mixture. The specificity and color variation of the substrates were further tailored to marine luciferases (or single-chain bioluminescent probes) to create a multiplex quadruple assay system with four integrated, single-chain bioluminescent probes, with each probe designed to selectively luminesce only with its specific ligand (first authentication) and a specific CTZ analogue (second authentication). This unique multiplex quadruple bioluminescent assay system is an efficient optical platform for specific and high-throughput imaging of multiple optical markers in bioassays without optical cross-leakages.

SUBMITTER: Kamiya G

PROVIDER: S-EPMC9581999 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Multiplex quadruple bioluminescent assay system.

Kamiya Genta G Kitada Nobuo N Maki Shojiro S Kim Sung Bae SB

Scientific reports 20221019 1

Bioluminescence (BL) is unique cold body radiation of light, generated by luciferin-luciferase reactions and commonly used in various bioassays and molecular imaging. However, most of the peak emissions of BL populate the blue-yellow region and have broad spectral bandwidths and thus superimpose each other, causing optical cross-leakages in multiplex assays. This study synthesized a new series of coelenterazine (CTZ) analogues, named K-series, that selectively illuminates marine luciferases with ...[more]

PMID: 36261452

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Project description:A sensitive multiplex PCR assay for single-tube amplification that detects simultaneous herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), varicella-zoster virus (VZV), human cytomegalovirus (CMV), and Epstein-Barr virus (EBV) is reported with particular emphasis on how the method was optimized and carried out and its sensitivity was compared to previously described assays. The assay has been used on a limited number of clinical samples and must be thoroughly evaluated in the clinical context. A total of 86 cerebrospinal fluid (CSF) specimens from patients which had the clinical symptoms of encephalitis, meningitis or meningoencephalitis were included in this study. The sensitivity of the multiplex PCR was determined to be 0.01 and 0.03 50% tissue culture infective doses/the reciprocal of the highest dilution positive by PCR for HSV-1 and HSV-2 respectively, whereas for VZV, CMV and EBV, 14, 18, and 160 ag of genomic DNA were detected corresponding to 48, 66, and 840 genome copies respectively. Overall, 9 (10.3%) of the CSF samples tested were positive in the multiplex PCR. HSV-1 was detected in three patients (3.5%) with encephalitis, VZV was detected in four patients (4.6%) with meningitis, HSV-2 was detected in one neonate (1.16%), and CMV was also detected in one neonate (1.16%). None of the samples tested was positive for the EBV genome. None of the nine positive CSF samples presented herpesvirus coinfection in the central nervous system. Failure of DNA extraction or failure to remove any inhibitors of DNA amplification from CSF samples was avoided by the inclusion in the present multiplex PCR assay of alpha-tubulin primers. The present multiplex PCR assay detects simultaneously five different herpesviruses and sample suitability for PCR in a single amplification round of 40 cycles with an excellent sensitivity and can, therefore, provide an early, rapid, reliable noninvasive diagnostic tool allowing the application of antiviral therapy on the basis of a specific viral diagnosis. The results of this preliminary study should prompt a more exhaustive analysis of the clinical value of the present multiplex PCR assay.

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Multiplex quadruple bioluminescent assay system.

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Multiplex quadruple bioluminescent assay system.

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