Recombinase polymerase amplification as a promising tool in hepatitis C virus diagnosis

Table 1 Characters of some isothermal amplification techniques[51]

	NASBA	LAMP	SDA	RCA	HDA	RPA
Template	DNA, RNA	DNA1	DNA1	DNA1	DNA1	DNA1
No. of primers	2	4-6	4	1	2	2
No. of enzymes	3	1	2	2	2	2
Temperature (°C)	41	60-65	37	37	65	30-42
Reaction duration (min)	90-120	60-90	120	60	75-90	20
Denaturation step	Y	N	Y	N	N	N
Inhibition tolerance	N	Y	N	N	Y	Y
Product detection	GE, RT	GE, RT, TE	GE, RT	GE	GE, RT	RT
Multiplex	Y	N	Y	N	Y	Y
Point-of-care	Y	Y	Y	N	Y	Y

¹RNA can be amplified after the introduction of a reverse transcription step. NASBA: Nucleic acid sequence-based amplification; LAMP: Loop-mediated isothermal amplification; SDA: Strand-displacement amplification; RCA: Rolling circle amplification; HDA: Helicase-dependent amplification; RPA: Recombinase polymerase amplification; GE: Gel Electrophoreses; RT: Real Time; TE: Turbidity; Y: Yes; N: No.

Table 2 Advantages/disadvantages of some isothermal amplification methods

Technique	Advantages	Disadvantages
NASBA	Specifically designed to detect RNA and in turn RNA viruses	Denaturation step
	Power saving (41 °C)	Less efficient in Amplifying RNA targets out of the range 120-250 bp
LAMP	Highly specific (utilizes 4-6 primers spanning 6-8 distinct sequences)	Primer design is complex
	Tolerance to biological substances	Unable to perform multiplex amplification
	Could be detected by a cheap turbidity-meter
SDA	Power saving (37 °C)	Sample prep. needed
		Nuclease selection is complex
		Inefficient in long target sequences
RCA	Power saving (37 °C)	Primer is complex
	Specific enough to allow SNP analysis	RNA amplification is complex
		Works only with a circular nucleic acid template
HDA	Simple primer design	Expensive enzymes
	Robust to biological substances
	No initial heating step
RPA	Power saving (37 °C)
	Simple primer design
	Extremely quick (20 min)
	No initial heating step
	Robust to biological substances

NASBA: Nucleic acid sequence-based amplification; LAMP: Loop-mediated isothermal amplification; SDA: Strand-displacement amplification; RCA: Rolling circle amplification; HDA: Helicase-dependent amplification; RPA: Recombinase polymerase amplification.