Development of a point-of-care test to detect hepatitis B virus DNA threshold relevant for treatment indication

Saran Pankaew; Sittiporn Pataradilokrat; Jantana Kampeera; Wansika Kiatpathomchai; Kiat Ruxrungtham; Siwaporn Boonyasuppayakorn

doi:10.1515/abm-2019-0021

.blurhash-client-img { display: none !important; }

Development of a point-of-care test to detect hepatitis B virus DNA threshold relevant for treatment indication

Asian Biomedicine

Volume 12 (2018): Issue 5 (October 2018)

By: Saran Pankaew, Sittiporn Pataradilokrat, Jantana Kampeera, Wansika Kiatpathomchai, Kiat Ruxrungtham and Siwaporn Boonyasuppayakorn

Open Access

|Oct 2019

Figures & Tables

Figure 1 HBV LAMP primer tests (A) Three newly designed HBV LAMP primer sets (S1, S2, and X) and a previous report [19] were tested with standard HBV DNA (108 copies/reaction) and deionized distilled water as positive and negative controls. (B) S2 primer set was tested with serially diluted standard HBV DNA (102–108 copies/reaction). M and NTC were abbreviated from marker and no template control, respectively. (C) Sensitivity results from polymerase chain reaction using universal primers.

Turbidity-based HBV LAMP sensitivity test S2 primer set was tested with serially diluted standard HBV DNA (102–108 copies/reaction) in (A) turbidimeter and (B) direct visualization at the end point of the reaction.

Specificity test S2 primer set was tested with 2 × 105 copies of standard DNAs of hepatitis B virus (HBV), human immunodeficiency virus (HIV), Epstein–Barr virus (EBV), cytomegalovirus (CMV), hepatitis C virus (HCV), and herpes simplex virus (HSV) and analyzed by 2% agarose gel electrophoresis.

Performance of turbidity-, and gel electrophoresis-based HBV LAMP assay at other cutoff titers of HBV qPCR results

Cutoff	Sensitivity		Specificity		PPV		NPV		Accuracy
Turbidity
10⁶	131/139	94.2%	74/105	70.5%	131/162	80.9%	74/82	90.2%	84.0%
5 × 10⁵	140/152	92.1%	70/92	76.1%	140/162	86.4%	70/82	85.4%	86.1%
10⁵	146/162	90.1%	66/82	80.5%	146/162	90.1%	66/82	80.5%	86.9%
5 × 10⁴	150/170	88.2%	62/72	83.8%	150/162	92.6%	62/82	75.6%	86.9%
10⁴	156/185	84.3%	53/59	89.8%	156/162	96.3%	53/82	64.6%	85.7%
10³	157/202	77.7%	37/42	88.1%	157/162	96.9%	37/82	45.1%	79.5%

Gel electrophoresis
10⁶	135/139	97.1%	63/105	60.0%	135/177	76.3%	63/67	94.0%	81.1%
5 × 10⁵	147/152	96.7%	62/92	67.4%	147/177	83.1%	62/67	92.5%	85.7%
10⁵	154/162	95.1%	59/82	72.0%	154/177	87.0%	59/67	88.1%	87.3%
5 × 10⁴	157/170	92.4%	54/67	73.0%	157/177	88.7%	54/67	80.6%	86.5%
10⁴	163/185	88.1%	45/59	76.3%	163/177	92.1%	45/67	67.2%	85.2%
10³	168/202	83.2%	33/42	78.6%	168/177	94.9%	33/67	49.3%	82.4%

Sensitivity of turbidity- and gel electrophoresis-based HBV LAMP detection system

HBV DNA (copies/ reaction)	Percent detection (No. of times detected/total replicated)
	Turbidity based	Gel electrophoresis based
2000	100 (6/6)	100 (3/3)
1000	100 (6/6)	100 (3/3)
200	83 (5/6)	100 (3/3)
150	83 (5/6)	100 (3/3)
100	100 (9/9)	100 (3/3)
50	50 (3/6)	67 (2/3)
20	67 (4/6)	67 (2/3)
10	33 (1/3)	–
5	33 (1/3)	–
2.5	67 (2/3)	–
1	33 (1/3)	–
0.5	0 (0/3)	–
0.1	33 (1/3)	–

Performance of turbidity-, and gel electrophoresis-based HBV LAMP assay at the 2 × 105 IU/mL cutoff titer of HBV qPCR results

Methods	Sensitivity (%)	Specificity (%)	Positive predictive value (%)	Negative predictive value (%)	Accuracy (%)
Turbidity-based	90.12 (146/162)	83.33 (90/108)	89.02 (146/164)	84.90 (90/106)	87.40 (236/270)
Gel electrophoresis	95.06 (154/162)	75.00 (81/108)	85.08 (154/181)	91.01 (81/89)	87.03 (235/270)

Cost comparison between quantitative PCR and LAMP

Methods	Price (per equipment)	Price (per reaction)			Price (per test, include positive and negative controls)

	Equipment	DNA extraction	Amplification reagents	Detection	Total
Quantitative PCR	qPCR 30,000–50,000 USD	5 USD	30 USD	–	105 USD

LAMP	Turbidimeter 2000 USD, or heat block (200–1200 USD), or waterbath (50–100 USD)	–	20 USD	–	60 USD

Oligonucleotide sequences of three newly designed and one previously reported primer sets

Primer set	Primer name	Location	Primer sequence (5‘ à 3‘)	Genetic region
S1	S1_F3	470–491	CCGTTTGTCCTCTAATTCCAGG	S and P gene
	S1_B3	665–688	GCACTAGTAAACTGAGCCAGGAGA
	S1_FIP	541–564	GGAGGGATACATAGAGGTTCCTTG-TTTT-CCTCAACAACCAGCACGGGA
		494–513
	S1_BIP	571–592	TGTACCAAACCTTCGGACGGAA-TTTT-CCCACTCCCATAGGAATTTTCC
		631–652
	S1_LF	520–540	AGCAGTAGTCATGCAGGTCCG
	S1_LB	595–616	TGCACCTGTATTCCCATCCCAT

S2	S2_F3	659–678	CTGCATGACTACTGCTCAAGGA	S and P gene
	S2_B3	712–731	AGCCAAACAGTGGGGGAAAG
	S2_FIP	595–615	TGGGATGGGAATACAGGTGCA-TTTT-CCTCTATGTATCCCTCCTGTTGCT
		548–571
	S2_BIP	631–651	GGAAAATTCCTATGGGAGTGGG-TTTT-CCCTACGAACCACTGAACAAATGG
		688–711
	S2_LF	572–591	TCCGTCCGAAGGTTTGGTAC
	S2_LB	659–678	CCCGTTTCTCCTGGCTCAGT

X	X_F3	1493–1512	CCTTCTCCGTCTGCCGTTCC	X gene
	X_B3	1728–1747	CCCCAACTCCTCCCAGTCTT
	X_FIP	1577–1596	GTGAAGCGAAGTGCACACGG-TTTT-CACCTCTCTTTACGCGGACTCC
		1529–1540
	X_BIP	1627–1646	CGCCCACCAAATATTGCCAA-TTTT-TATGCCTCAAGGTCGGTCGTTG
		1686–1707
	X_LF	1559–1576	TCCGGCAGATGAGAAGGC
	X_LB	1662–1685	GGACTCTTGGACTCTCAGCAATGT

Nyan et al. [19]	Ref_F3	530–249	TCCTCACAATACCGCAGAGT	S and P gene
	Ref_B3	402–421	GCAGCAGGATGAAGAGGAAT
	Ref_FIP	305–326	GTTGGGGACTGCGAATTTTGGC-TTTT-TAGACTCGTGGTGGACTTCT
		251–270
	Ref_BIP	333–354	TCACTCACCAACCTCCTGTCCT-TTTT-AAAACGCCGCAGACACAT
		379–396
	Ref_LF	271–294	GGTGATCCCCCTAGAAAATTGAG
	Ref_LB	357–378	AATTTGTCCTGGTTATCGCTGG

References

Authors

Metrics

Articles in this issue

DOI: https://doi.org/10.1515/abm-2019-0021 | Journal eISSN: 1875-855X | Journal ISSN: 1905-7415

Journal RSS Feed

Language: English

Page range: 201 - 209

Published on: Oct 1, 2019

Published by: Chulalongkorn University

In partnership with: Paradigm Publishing Services

Publication frequency: 6 issues per year

Keywords:

Related subjects:

Assistive professions, nursing,

Basic medical science,

Basic medical science, other,

Clinical medicine,

Clinical medicine, other

© 2019 Saran Pankaew, Sittiporn Pataradilokrat, Jantana Kampeera, Wansika Kiatpathomchai, Kiat Ruxrungtham, Siwaporn Boonyasuppayakorn, published by Chulalongkorn University
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

Volume 12 (2018): Issue 5 (October 2018)

Development of a point-of-care test to detect hepatitis B virus DNA threshold relevant for treatment indication

Figures & Tables

Figure 1

Figure 2

Figure 3

Performance of turbidity-, and gel electrophoresis-based HBV LAMP assay at other cutoff titers of HBV qPCR results

Sensitivity of turbidity- and gel electrophoresis-based HBV LAMP detection system

Performance of turbidity-, and gel electrophoresis-based HBV LAMP assay at the 2 × 105 IU/mL cutoff titer of HBV qPCR results

Cost comparison between quantitative PCR and LAMP

Oligonucleotide sequences of three newly designed and one previously reported primer sets

Paradigm

My account