Progress in Modern Biomedicine Vol.11 NO.7 APR ' 10 min PCR 250 copies/ml PCR

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1 1277 HBV DNA* ' 10 min HBsAg PCR 250 copies/ml PCR 100% PCR DNA R446.5 B Development of a sensitive, visual nucleic acid dipstick assay for rapid detection of hepatitis B virus DNA* YANG Xian 1, HUANG Huan 2, YIN Zhu-jun 3, WU Hai-ping 2, ZHOU Guo-hua 1 (1 School of Basic Medical Sciences, Nanjing Medical University, , Nanjing, China; 2 Huadong Research Institute for Medicine and Biotechnology, , Nanjing, China; 3 Jiangsu Entry-Exit Inspection and Quarantine Bureau, , Nanjing, China) ABSTRACT Objective: We aimed to develop a nucleic acid dipstick assay (NADA) for rapid and visual detection of Hepatitis B virus (HBV) with a high specificity and sensitivity. Methods: PCR amplification of HBV conserved sequences was carried out by a pair of primers labeled with FITC and biotin at the 5' end respectively. The gold nanoparticles and test zones in the dipstick were modified with anti-fluorescein and strepavidin respectively. PCR products mixed with developing buffer were applied to the sample-loading area of the dipstick and the detection result can be observed 10 min afterward with naked eyes. As the buffer migrates along the strip by capillary action, the PCR products were captured at the test zones by immobilized anti-fluorescein and detected by strepavidin-funtcionalized gold nanoparticles, thus generating characteristic red lines. The excess nanoparticles were captured by immobilized biotinylated albumin at the control zone of the strip and then form another red line. We have optimized the developing buffer as well as the volume and concentration for sample-loading. The sensitivity of the optimized NADA has also been evaluated. To demonstrate the utility of NADA, a total of 48 clinical samples containing 15 negative control and 33 HBsAg positive cases were detected. The results from NADA were further compared with that from qpcr. Results: The sensitivity of NADA for visual detection of HBV was 250 copies/ml. In the clinical tests, the specificity of DADA and qpcr was 100%, respectively. There were no differences for the detection rates of discriminating the different serum-marker patterns by NADA and qpcr. Conclusions: Our newly developed NADA could be applied for visual detection of HBV. Compared with qpcr, NADA have the advantages of the rapidity, higher sensitivity and specificity, holding a great promise for wide applications in epidemiological surveys and routine physical examinations in hospitals. Key words: Nucleic acid dipstick assay; Polymerase chain reaction; Hepatitis B virus DNA Chinese Library Classification: R446.5 Document code: B Article ID: (2011) (Hepatitis B virus, HBV) * (BK ) ( ) (1985-) yangxian-gp4@163.com : ghzhou@nju.edu.cn

2 [1] 6 μl 94 μl (PBS PCR(Quantitative Real-Time PCR, qpcr) ) 10 min T + 0.1% BSA, ph 7.4) 100 μl ( [2, 3] qpcr, T C 1.5 (Gold Immunochromagraphy Assay, GICA) "GeneTools from Syngene" [4] 2,, GICA 10 min (Nucleic acid dipstick assay, NADA) 5' (F) (B) 10 min (SA) 1 (Anti-F) (B) (HBsAg) Taq DNA PCR TaKaRa Invitrogen (HBV) (HBV) PCR (EDC-810 ) ; PCR DNA Engine Opticon 2 (POWPAC 200) Bio-Rad Syngene Naka Instrument (Pico17) Thermo Fisher Scientific NCBI S Clustal X 5' 1 5' Primer Premier Fig.1 Schematic illustration of the nucleic acid dipstick assay. S: sample application area; T: test zone for HBV; C: control zone; F: fluorescein isothiocyanate; B: biotin; Au: gold nanoparticles; SA: streptavidin; Anti-F: 1.2 anti-fluorescein; BSA: bovine serum albumin. 100 μl (HBV) 10 μl μl μl 10 PCR Buffer 1.5 μl MgCl 2 (25mM) 0.5 μl dntp Mix (10 mm) 1 μl (10 pmol/μl) Taq DNA μl (5 U/μL) H 2 O 25 μl : 94 2 min 90 10s 70 15s 72 15s min Syngene (Cross dimers, CDs) FITC-P1/ Biotin-P2 ( 1) 37

1279 1 h, ΔG 3 ( 3 CDs ) Tm Taq 3 PC FITC-P3/ Biotin-P4 ( 1) ΔG =6.

4 Biotin-P2 Biotin-GGTCTTCCAAATTACTTCCCACCCA 66.3 FITC-P3 FITC-GCAGTCCCCAACCTCCAATCAC 66.9 Biotin-P4 Biotin-GAAGTAGAGGACAAACGGGCAACATA 66.1 0.01 mol/l PBS( ) ( ph 7.4) PBS + 0.1% BSA( ) PBS + 0.

3 h, ΔG 3 ( 3 CDs ) Tm Taq 3 PC FITC-P3/ Biotin-P4 ( 1) ΔG =6.2 Tm CDs PCR 3 1 3' 2 3' 1-4 ( ΔG ) 1 Table 1 Primer sequences used in this study Primer Sequences (5' 3') Tm ( ) FITC-P1 FITC-GATCTCCTCGACACCGCCTCAG 67.4 Biotin-P2 Biotin-GGTCTTCCAAATTACTTCCCACCCA 66.3 FITC-P3 FITC-GCAGTCCCCAACCTCCAATCAC 66.9 Biotin-P4 Biotin-GAAGTAGAGGACAAACGGGCAACATA mol/l PBS( ) ( ph 7.4) PBS + 0.1% BSA( ) PBS + 0.1% Tween 20 Tris EDTA (TE, ph 8.0) 96 μl 4 μl (1010 copies/μl) PBS % BSA ( 5a) BSA - 2 ( ) Fig.2 Electrophoregrams (up) and dipstick-images (down) of cross dimers at different PCR thermal cycling conditions. 1: 30 cycles; 2: 35 cycles; 3: 40 cycles; 4: 45 cycles; CDs: Cross dimers; PC: Positive control; M: Maker. 2.3 / / ( 4 1) ( 4 2) ( 4 3) ( 4 4) 3 / 4 Fig.3 Evaluation of specificity of antibody/hapten pairs. 1: P3/ P4; 2: FITC-P3/ P4; 3: P3/ Biotin-P4; 4: FITC-P3/ Biotin-P4. / μl 2 μl 4 μl 6 μl 8 μl (1010 copies/μl)

1280 100 μl 250 copies/ml 500 copies/ml 6 μl ( 5b) 2.4.3 1011 copies/μl 10 100 109 copies /μl PCR 85%, [5] 1010 copies/μl~1011 copies/μl (NC) (1010 4 copies/μl) 1.9 % Fig.

(b) Effect of sample-loading volume on the signal intensity of PCR the test zone in the dipstick.

4 μl 250 copies/ml 500 copies/ml 6 μl ( 5b) copies/μl copies /μl PCR 85%, [5] 1010 copies/μl~1011 copies/μl (NC) ( copies/μl) 1.9 % Fig.4 Optimization of the conditions in nucleic acid dipstick assay. PCR (a) Effect of buffer components on the signal intensity of the test zone in 2.5 the dipstick. (b) Effect of sample-loading volume on the signal intensity of PCR the test zone in the dipstick. (c) Evaluation of the sensitivity using different copies/ml amount of PCR amplicons as the loading sample. TE: Tris EDTA; NC: 6 Negative control. 5 PCR Fig.5 The sensitivity of nucleic acid dipstick coupled with PCR amplification for HBV detection. NC: Negative control C [9] HBsAg HBV-DNA 2 PCR IV V qpcr 100 % 2 15 ( ) 3 HBV qpcr 88.2 % 2 HBV DNA 7 8 %~15 % [6] qpcr [7,8] C/C X S S 60, 90 PCR " " 70 Taq PCR 72.7 % " 90 PCR 45 min " 10 min 60 " " HBV-DNA 11 PCR 30 min

5 1281 / copies/ml [10] 500 μl copies/ml 250 copies/ml HBV DNA 50 copies/ml ~ Table 2 Detection results of different clinical samples by NADA and qpcr Sample groups HBV serum-markers No. of samples (%) detected by: No. of samples HBsAg HBsAb HBeAg HBeAb HBcAb NADA qpcr (100) 15 (100) (88.2) 15 (88.2) (72.7) 8 (72.7) (100) 4 (100) (100) 1 (100) A-gold complexes [J]. Anal Biochem, 1984, 142(1): [5] Saiki RK, Scharf S, Faloona F, et al. Enzymatic amplification of betaglobin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia [J]. Science, 1985, 230(4732): (References) [6] Kidd-Ljunggren K, Miyakawa Y, Kidd AH. Genetic variability in hepatitis B viruses [J]. J Gen Virol, 2002, 83(6): [1],. [J]., 2011,1: Chinese Society of Hepatology and Chinese Society of Infectious Diseases, Chinese Medical Association. The guideline of prevention and treatment for chronic hepatitis B, Journal of Pathogen Biology [J]. 2011, 1: 67-77(In Chinese) [2] Guirgis BS, Abbas RO, Azzazy HM. Hepatitis B virus genotyping: current [7] Kao JH, Chen PJ, Lai MY, et al. Sequence analysis of pre-s/surface and pre-core/core promoter genes of hepatitis B virus in chronic hepatitis C patients with occult HBV infection [J]. J Med Virol, 2002, 68 (2): [8] De Mitri MS, Morsica G, Cassini R, et al. Low replication and variability of HBV pre-core in concomitant infection with hepatitis B and methods and clinical implications [J]. Int J Infect Dis, 2010, 14 hepatitis C viruses [J]. Arch Virol, 2007, 152(2): (11): e941-e953 [9] De Mitri MS, Cassini R, Morsica G, et al. Virological analysis, genotypes [3] Chevaliez S, Bouvier-Alias M, Laperche S, et al. Performance of version 2.0 of the Cobas AmpliPrep/Cobas TaqMan real-time PCR assay and mutational patterns of the HBV precore/core gene in HBV/HCV-related hepatocellular carcinoma [J]. J Viral Hepat, 2006, for hepatitis B virus DNA quantification [J]. J Clin Microbiol, 2010, 13(9): (10): [10] Biswas R, Tabor E, Hsia CC, et al. Comparative sensitivity of HBV [4] Brada D, Roth J. "Golden blot"--detection of polyclonal and monoclonal antibodies bound to antigens on nitrocellulose by protein NATs and HBsAg assays for detection of acute HBV infec tion [J]. Transfusion, 2003, 43(6):

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