PMID- 34049623
OWN - NLM
STAT- MEDLINE
DCOM- 20210603
LR  - 20210603
IS  - 1873-4324 (Electronic)
IS  - 0003-2670 (Linking)
VI  - 1167
DP  - 2021 Jul 4
TI  - Accurate nucleic acid quantification in a single sample tube without the need for 
      calibration.
PG  - 338599
LID - S0003-2670(21)00425-6 [pii]
LID - 10.1016/j.aca.2021.338599 [doi]
AB  - Convenient and accurate nucleic acid quantification (NAQ) is crucial to clinical 
      diagnosis, forensic medicine, veterinary medicine and food analysis. However, 
      traditional NAQ relies on the preparation of a laborious, time-consuming and 
      expensive calibration curve, which would also propagate pipette errors through 
      serially dilutions. Besides, traditional NAQ is run in different tubes, which 
      introduces bias from random tube-to-tube variations and is unable to detect 
      inhibitors from biological samples. To solve these problems, a single-tube 
      quantitative PCR (stqPCR) technique is proposed which enables accurate 
      quantification without the need for a calibration curve. In this method, an 
      internal quantitative standard DNA (IQS-DNA) for quantification was screened out 
      by co-amplification with the target DNA. Then the difference between the 
      quantification cycle value (DeltaCq) of the IQS-DNA and the target DNA was used for 
      NAQ. The method permitted high accuracy quantification with reliable data for 
      concentrations in plasmid, serum standard, and clinical samples being confirmed 
      (R(2) values of 0.9951, 0.9889, and 0.9727, slope values of 1.011, 1.028, and 
      0.9327, and intercept values of -0.06037, -0.1486, and 0.3325, respectively). 
      Accurate NAQ could also be achieved by stqPCR even though inhibitors were present 
      in a sample; however, in the case of using a commercial assay kit, satisfactory 
      performance was only attained after the same sample was diluted some 32-fold. 
      Moreover, integration of the present method into a microfluidic system could 
      achieve super-fast NAQ in less than 30 min and achieve super-fast "sample in, 
      quantitative answer out" testing in less than 40 min. Thus, the stqPCR method 
      present here would promote the development of NAQ in the laboratory and on site.
CI  - Copyright (c) 2021 Elsevier B.V. All rights reserved.
FAU - Xu, Jiasu
AU  - Xu J
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Wang, Jin
AU  - Wang J
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China; School of Life 
      Sciences, Xiamen University, Xiamen, 361102, China.
FAU - He, Shuizhen
AU  - He S
AD  - Haicang Hospital of Xiamen, Xiamen, 361026, China.
FAU - Su, Xiaosong
AU  - Su X
AD  - Xiang'an Hospital of Xiamen University, Xiamen, 361102, China.
FAU - Zhong, Zecheng
AU  - Zhong Z
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Zhong, Weibo
AU  - Zhong W
AD  - Xiamen Innovax Biotech CO., LTD., Xiamen, 361022, China.
FAU - Yan, Lizhen
AU  - Yan L
AD  - Haicang Hospital of Xiamen, Xiamen, 361026, China.
FAU - Huang, Shaolei
AU  - Huang S
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Yang, Jiayu
AU  - Yang J
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Gao, Runxin
AU  - Gao R
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Zhang, Jianbin
AU  - Zhang J
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Zeng, Juntian
AU  - Zeng J
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Zhang, Dongxu
AU  - Zhang D
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Li, Tingdong
AU  - Li T
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Zhang, Shiyin
AU  - Zhang S
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China. Electronic address: 
      zhangshiyin@xmu.edu.cn.
FAU - Ge, Shengxiang
AU  - Ge S
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China. Electronic address: 
      sxge@xmu.edu.cn.
FAU - Zhang, Jun
AU  - Zhang J
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China.
FAU - Xia, Ningshao
AU  - Xia N
AD  - State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, National 
      Institute of Diagnostics and Vaccine Development in Infectious Diseases, School 
      of Public Health, Xiamen University, Xiamen, 361102, China; School of Life 
      Sciences, Xiamen University, Xiamen, 361102, China.
LA  - eng
PT  - Journal Article
DEP - 20210503
PL  - Netherlands
TA  - Anal Chim Acta
JT  - Analytica chimica acta
JID - 0370534
RN  - 9007-49-2 (DNA)
SB  - IM
MH  - Calibration
MH  - *DNA
MH  - *Food Analysis
MH  - Real-Time Polymerase Chain Reaction
OTO - NOTNLM
OT  - Inhibitor-tolerant
OT  - Microfluidics
OT  - Nucleic acid quantification
OT  - Single sample tube
OT  - Without calibration curve
COIS- Declaration of competing interest The authors declare that they have no known 
      competing financial interests or personal relationships that could have appeared 
      to influence the work reported in this paper.
EDAT- 2021/05/30 06:00
MHDA- 2021/06/04 06:00
CRDT- 2021/05/29 05:30
PHST- 2021/01/14 00:00 [received]
PHST- 2021/04/14 00:00 [revised]
PHST- 2021/04/28 00:00 [accepted]
PHST- 2021/05/29 05:30 [entrez]
PHST- 2021/05/30 06:00 [pubmed]
PHST- 2021/06/04 06:00 [medline]
AID - S0003-2670(21)00425-6 [pii]
AID - 10.1016/j.aca.2021.338599 [doi]
PST - ppublish
SO  - Anal Chim Acta. 2021 Jul 4;1167:338599. doi: 10.1016/j.aca.2021.338599. Epub 2021 
      May 3.