A comparative study of extraction free detection of HBV DNA using sodium dodecyl sulfate, N-lauroylsarcosine sodium salt, and sodium dodecyl benzene sulfonate

被引：0
|
作者：
Ko, Ko [1 ,2 ]
Lokteva, Lyubov Mikhailovna [1 ,3 ]
Akuffo, Golda Ataa [1 ,2 ]
Phyo, Zayar [1 ,2 ]
Chhoung, Chanroth [1 ,2 ]
Bunthen, E. [1 ,4 ]
Ouoba, Serge [1 ,5 ]
Sugiyama, Aya [1 ,2 ]
Akita, Tomoyuki [1 ,2 ]
Rattana, Kim [6 ]
Vichit, Ork [7 ]
Takahashi, Kazuaki [1 ,2 ]
Tanaka, Junko [1 ,2 ]
机构：
[1] Hiroshima Univ, Grad Sch Biomed & Hlth Sci, Dept Epidemiol Infect Dis Control & Prevent, 1-2-3 Kasumi, Minami, Hiroshima 7348551, Japan
[2] Hiroshima Univ, Project Res Ctr Epidemiol & Prevent Viral Hepatiti, Hiroshima, Japan
[3] Res Inst Virol, Tashkent, Uzbekistan
[4] Minist Econ & Finance, Natl Social Protect Council, Natl Payment Certificat Agcy, Phnom Penh, Cambodia
[5] Inst Rech Sci Sante IRSS, Unite Rech Clin Nanoro URCN, Nanoro, Burkina Faso
[6] Natl Maternal & Child Hlth Ctr NMCHC, Minist Hlth, Phnom Penh, Cambodia
[7] Minist Hlth, Natl Immunizat Program NIP, Phnom Penh, Cambodia
来源：
SCIENTIFIC REPORTS | 2024年 / 14卷 / 01期
基金：
日本学术振兴会;
关键词：
HBV DNA; qPCR; Surfactant; Sodium dodecyl sulfate; N-Lauroylsarcosine sodium salt; Sodium dodecyl benzene sulfonate; POLYMERASE-CHAIN-REACTION; HEPATITIS-B; MOLECULAR DIAGNOSTICS; VIRUS-DNA; PCR; PROTEIN; AMPLIFICATION; PURIFICATION; EXPRESSION;
D O I：
10.1038/s41598-024-75944-7
中图分类号：
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号：
07 ; 0710 ; 09 ;
摘要：
This study aimed to develop an extraction-free method for quantitative and qualitative detection of HBV DNA compared to traditional nucleic acid extraction. Paired serum and dried blood spot (DBS) samples from 67 HBsAg-positive and 67 HBsAg-negative individuals were included. Two samples with known HBV DNA titers (similar to 109 copies/mL) were examined by extraction-free detection using three surfactants (0.2 to 1% of Sodium dodecyl sulfate:SDS, N-Lauroylsarcosine sodium salt:NL, Sodium dodecyl benzene sulfonate:SDBS), two stabilizing agents (0.1 or 0.01% 2-Mercaptoethanol:2ME and 3.5 or 7% Bovine Serum Albumin:BSA) and two Taq polymerases (Fast Advanced and Prime Direct Probe). HBV DNA in all 67 HBsAg-positive and 67 HBsAg-negative serum and DBS samples was directly quantified by Rt-PCR using 0.4% SDS or 0.4% NL with Fast Advance or Prime Direct Probe Taq. Extraction-free amplification was also performed. Detection limits were varied by different surfactants and Taq. SDS combined with Fast Advanced Taq showed lower detection limits, while SDS with Prime Direct Probe Taq outperformed NL or SDBS-based detection. Adding 2ME to SDS improved detection limit with Prime Direct Probe Taq but not significantly compared to SDS alone. BSA did not significantly enhance detection limits but provided insights into sample stability. The senitivity and specificity of 0.4% SDS and NL in combination with either Fast advanced or Prime Direct Probe Taq polymerase in serum samples were > 90% and 100% resepctively, while it was > 80% and 100% respectively in DBS samples. Extraction-free HBV DNA amplification provided 100% identity with original genomes. Our study suggests that SDS, NL or SDBS-based extraction-free HBV DNA detection strategies using Prime Direct Probe Taq have potential to simplify and accelerate HBV DNA detection with high sensitivity and specificity in both serum and DBS samples, with implications for resource-limited settings and clinical applications. Utilizing surfactants with 2ME is optional, and further research and validation are necessary to broaden its application in real-world diagnostics.
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页数：13
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