Simple and feasible detection of hepatitis a virus using reverse transcription multienzyme isothermal rapid amplification and lateral flow dipsticks without standard PCR laboratory

Figures & data

Figure 1. The primer targeting regions.

Table 1. The primers used for MIRA.

Primer	Sequence (5’ to 3’)	Position in HAV sequence*	Product length (bp)
F-a1	AGGGGACTTGATACCTCACCGCCGTTTG	53–80	180
R-a1	TGGAAGAAAGAAGACAGAAAGCGTGAAAAATGAG	199–232
F-a2	CACTCATTTTTCACGCTTTCTGTCTTCTTTC	197–227	269
R-a2	CCGCCGCTGTTACCCTATCCAAGGCATCT	437–465
F-a1 labelled	[5′-FAM]-AGGGGACTTGATACCTCACCGCCGTTTG	53–80	180
R-a1 labelled	[5′-biotin]-TGGAAGAAAGAAGACAGAAAGCGTGAAAAATGAG	199–227

*NCBI Reference Sequence: NC_001489.1.

Table 2. The primers used for RT-MIRA of HAV vaccine strain H2.

Primer	Sequence (5’ to 3’)	Position in H2 stain sequence	Product length (bp)
F-a1A	AGGGGACTTGATACCTCACCGCCGTTTG	53–80	175
R-a1A	TGGAAGAAAGAAGACAGAAAGCGTGAAAATGAG	195–227
F-a1A labelled	[5′-FAM]-AGGGGACTTGATACCTCACCGCCGTTTG	53–80	175
R-a1A labelled	[5′-biotin]-TGGAAGAAAGAAGACAGAAAGCGTGAAAATGAG	195–227

Figure 2. MIRA products separated on a 2% agarose gel. L: 50-bp DNA Ladder; 1: positive control (∼250 bp); 2: primer a1 (∼180 bp); 3: primer a2 (∼269 bp); 4: primer a1, negative control; 5: primer a2, negative control.

Figure 3. The MIRA products for the serial dilution of HAV plasmid DNA separated on a 2% agarose gel. L: 50-bp DNA Ladder; 1: positive control (∼250 bp); 2: 10⁷ copies/μl template with a1 primers; 3: 10⁶ copies/μl template with a1 primers; 4: 10⁵ copies/μl template with a1 primers; 5: 10⁴ copies/μl template with a1 primers; 6: 10⁷ copies/μl template with a2 primers; 7: 10⁶ copies/μl template with a2 primers; 8: 10⁵ copies/μl template with a2 primers; 9: 10⁴ copies/μl template with a2 primers.

Figure 4. The Sanger sequence of the HAV plasmid DNA product generated using primer a1.

Figure 5. Cross-reactivity testing against multiple pathogens using MIRA. L: 50-bp DNA Ladder; 1: positive control (∼250 bp); 2: HAV; 3: HBV; 4: HCV; 5: HEV; 6: HIV-1; 7: HSV-1; 8: JEV; 9: rotavirus 10: human genome.

Figure 6. Serial dilution of HAV plasmid DNA detected by MIRA-LFD. 1: water template; 2: human genome template; 3: 10⁶ copies/μl template; 4: 10⁵ copies/μl template; 5: 10⁴ copies/μl template; 6: 10³ copies/μl template; 7: 10² copies/μl template; 8: 10 copies fg/μl template; 9: 1 copy/μl template; 10: 0.1 copy/μl template.

Figure 7. Serial dilution of HAV plasmid DNA detected by qPCR. 1: 10⁷ copies/μl template; 2: 10⁶ copies/μl template; 3: 10⁵ copies/μl template; 4: 10⁴ copies/μl template; 5: 10³ copies/μl template; 6: 10² copies/μl template; 7: 10 copies/μl template; 8: water template.

Figure 8. RT-MIRA products for the serial dilution of HAV vaccine strain H2 template separated on a 2% agarose gel. L: 50-bp DNA Ladder; 1: plasmid DNA template, positive control; 2: water template, negative control; 3: 10⁶ copies/μl template; 4: 10⁵ copies/μl template; 5: 10⁴ copies/μl template; 6: 10³ copies/μl template; 7: 10² copies/μl template

Figure 9. The Sanger sequence of the HAV vaccine strain H2 product generated by RT-MIRA.

Figure 10. Serial dilution of HAV vaccine strain H2 template detected by RT-MIRA-LFD. 1: water template; 2: human genome template; 3: 10⁵ copies/μl template; 4: 10⁴ copies/μl template; 5: 10³ copies/μl template; 6: 10² copies/μl template; 7: 10 copies/μl template; 8: 1 copy/μl template; 9: 0.1 copy/μl template.

Table 3. HAV detection in clinical samples.

		Real-time PCR			Accuracy rate
		positive	negative	total
MIRA-LFD	positive	19	0	19	100%
	negative	0	16	16
	total	19	0	35

Figure 11. Scheme summarising of the complete testing process.

Data availability statement

The data sets used and/or analysed during this study are available from the corresponding author on reasonable request.