A single-tube nucleotide isolation reagent for the quantitative PCR detection of virus in body fluids.
A high-salt reagent composed of guanidinium thiocyanate, guanidine hydrochloride, urea, sodium citrate, and other compounds was designed for the single-tube isolation of viral nucleotides from body fluids. The single-tube reagent was used for the extraction of SIV RNA and HBV DNA from standard virus stock dilutions and virus-positive samples. The sensitivity and reproducibility of the single-tube reagent were analysed via quantitative PCR assays. The results revealed that the single-tube reagent can facilitate quantitative PCR-mediated detection in a reaction system with a 25-μl volume using only 100 μl of a body fluid sample and reaches a sensitivity of up to 50 copies/ml. The low coefficients of variance of both the HBV and SIV standard stock results indicate the excellent reproducibility of the single-tube reagent. A Bland-Altman analysis of the assay results from the SIV- and HBV-positive samples revealed that the single-tube reagent can precisely extract both RNA and DNA viral nucleotides from virus-positive samples. All of the isolation steps were performed in a single tube and were completed in no more than 35 min. The only major equipment required is a high-speed freezing centrifuge. The single-tube reagent is economical and easy to use and does not require any complex equipment.
Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou 510405, China.
Hong
H
Liu
The Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou 510405, China.
Yuansheng
Y
Wu
Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou 510405, China.
Cuihua
C
Liu
Tropical Medicine Institute, Guangzhou University of Chinese Medicine, Guangzhou 510405, China. Electronic address: 303877469@qq.com.
Jinyang
J
He
D000818:Animals; D001826:Body Fluids / Q000821:virology*; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D006515:Hepatitis B virus / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000302:isolation & purification; D013048:Specimen Handling / Q000379:methods*
D004279:DNA, Viral; D012367:RNA, Viral
Isolation and purification; Nucleotides; Polymerase chain reaction; Viruses