Reverse transcription loop-mediated isothermal amplification for the detection of highly pathogenic porcine reproductive and respiratory syndrome virus.

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the open reading frames 1a of highly pathogenic porcine reproductive and respiratory syndrome virus genome was developed. The 10 reference strains, 1 clinical isolation strain and 122 positive samples were tested. Positive reactions were confirmed for all strains and specimens by reverse transcription loop-mediated isothermal amplification and nested reverse transcription polymerase chain reaction (RT-PCR). The results showed this detection technique is more reliable and convenient for rapid and sensitive diagnosis of highly pathogenic porcine reproductive and respiratory syndrome virus infection.

Porcine reproductive and respiratory syndrome (PRRS) is a serious swine disease and the causing agent is PRRS virus (PRRSV) which belongs to the member of arteriviruses, a group of small, enveloped, positive-strand RNA virus (Conzelmann et al., 1993) PRRS was first observed in the United States in 1987 (Keffaber, 1989) and in Europe in 1990 (Wensvoort et al., 1991). To date, PRRS has spread worldwide and caused enormous economic losses each year (Gao et al., 2004). Recently, the unparalleled large-scale outbreaks of a highly pathogenic PRRS, which spread to many provinces in China, have cause severe economic losses for the Chinese swine industry. Autopsies combined with immunological tests showed clearly that multiple organs were infected by the highly pathogenic PRRSV with severe pathological changes observed (Tian et al., 2007, Li et al., 2007, Normile, 2007). The prerequisite for controlling the disease is a rapid and accurate identification of this organism.

Virus isolation of PRRSV is difficult. This is mainly because the cell of choice for virus isolation is the porcine alveolar macrophage, which needs to be harvested from pigs (preferably specific pathogen free [SPF]) under 6–8 weeks of age (Wensvoort et al., 1991, Yoon et al., 1992). Not all laboratories have a ready supply of such pigs available, and continuous cell lines cannot replace fully the alveolar macrophages because these cell lines are generally less susceptible to the virus. In addition, different batches of macrophages are not always equally susceptible to the virus, and results are not obtained rapidly. Although reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive and specific method (Kono et al., 1996, Larochelle and Magar, 1997, Mardassi et al., 1994, Van Woensel et al., 1994), the dependence on special equipment limits its extensive use.

A novel nucleic acid amplification method, loop-mediated isothermal amplification (LAMP), relies on autocycling strand displacement DNA synthesis performed by Bst DNA polymerase (Notomi et al., 2000, Mori et al., 2001; Nagamine et al., 2002, Chen et al., 2008). Furthermore, reverse transcription LAMP (RT-LAMP) method has been applied successfully for the detection of human influenza A virus, severe acute respiratory syndrome coronavirus and Newcastle disease virus (Hong et al., 2004, Pham et al., 2005, Poon et al., 2005). In the present study, RT-LAMP method was developed with the HPBEDV strain for the detection of highly pathogenic PRRSV from blood, semen and lung samples. RNA transcripts corresponding to the open reading frames (ORF) 1a (nucleotides 2710–2946) of highly pathogenic PRRSV genome were generated to use as standards in the sensitivity analysis of the assay, respectively. A series of the five times dilutions spanning 1 to 55 copies/tube was used as template. Briefly, RNA was extracted from HPBEDV strain using the QIAamp RNA extraction kit. The purified RNA was resuspended in diethylpyrocarbonate treated water and used in the RT-PCR reaction. The amplified product of ORF 1a was cloned into the pCR-XL-TOPO vector (Invitrogen Inc., Shanghai, China) according to the manufacturer's directions and sequenced to verify its accuracy. The recombinant plasmid pCR-NSP2 was linearized and gel purified and used as template with a RiboMax T7 In Vitro Transcription System (Promega, Madison, WI) according to the manufacturer's recommendations. The length of the RNA transcripts was verified by agarose gel analysis, and the RNA of ORF 1a was quantitated using UV spectrophotometry at 260 nm. To test the applicability of this method, 10 reference strains and one clinical strain of highly pathogenic PRRSV (Table 1 ) were used. Strains were isolated from lung tissues of highly pathogenic PRRSV affected pigs and homogenised with Dulbecco's modified Eagles medium (DMEM), freeze–thawed three times and centrifuged at 10,000 ×  g for 10 min. The supernatant was passed through a 0.22-μm filter and adapted to Marc-145 cell monolayers. The cells were incubated at 37 °C for 5 days and examined for cytopathic effects (CPE) daily. After the appearance of CPE, viral isolates were stored at −70 °C until used. RNA was extracted by using a RNeasy Mini Kit (Qiagen). For further evaluation of RT-LAMP assay with clinical specimens, 122 specimens of blood, semen and lung tissue were obtained from highly pathogenic PRRSV-infected pigs (Table 2 ). The specimens were frozen at −70 °C until transported and tested.

Table 1

Strains used in this study

Strain	Genotype	Name of strain	GenBank access number
Reference strains	North America	CH-1a	AY032626
	North America	BJ-4	AF331831
	North America	HUB1	EF075945
	North America	HuN	EF517962
	North America	HUN4	EF635006
	North America	HN1	AY457635
	North America	JXwn06	EF641008
	North America	JX0612	EF488048
	North America	JXA1	EF112445
	North America	GD	EU109503
Clinical isolate	North America	HPBEDV	EU236259

Table 2

Comparative analysis of the highly pathogenic PRRSV specimens by RT-LAMP and nested RT-PCR

Specimen	Strain (positive number)	Positive number of result by
		RT-LAMP	Nested RT-PCR
Blood	JXA1 (12)	12	12
	GD (13)	13	13
	HPBEDV (12)	12	12
Semen	JXA1 (13)	13	13
	GD (12)	12	12
	HPBEDV (19)	19	19
Lung	HPBEDV (41)	41	41

Four primers of FIP, BIP, F, and B for the RT-LAMP test were designed by targeting the conserved regions of ORF 1a (GenBank access number EU236259) and listed in Table 3 . RT-LAMP was performed in 25 μL of a mixture containing 2 μL of the genomic RNA, 40 pmol (each) of primers FIP and BIP, 5 pmol (each) of primers F3 and B3, 1 U of the THERMO-X reverse transcriptase (Invitrogen) and 8 U of Bst DNA polymerase (New England Biolabs, Ipswich, MA) with the corresponding buffer, respectively. Amplification was carried out at 64 °C for 15, 30, 45, 60 and 75 min and electrophoresis analysis indicated that 45 min are enough for highly pathogenic PRRSV RT-LAMP in the study. The reaction was then terminated by incubation at 80 °C for 2 min. RT-LAMP products for highly pathogenic PRRSV and PRRSV-infected blood, semen and lung samples were analyzed by electrophoresis with 2.5% agarose gels (Fig. 1A). All the strains tested by RT-LAMP were also identified by nested RT-PCR and sequenced. The details of primers and condition for nested RT-PCR assay for the detection of PRRSV have been previously described (Christopher-Hennings et al., 1995), with minor modifications. The outer sense and antisense primers were N1F and N1R and the nested sense and antisense primers were N2F and N2R, respectively(Table 3). A good correlation was found for all of the highly pathogenic PRRSV strains which were positive by the RT-LAMP and nested RT-PCR.

Table 3

Details of RT-LAMP and nested RT-PCR primers designed for the detection of highly pathogenic PPRSV

Method	Target	Primer	Genome positiona	Sequence
RT-LAMP	ORF1a	F	2710–2719	5′-GCTCCGCGCAGGAAGGTCA-3′
		B	2928–2946	5′-GTGCGTCAGCGTTGTTGTC-3′
		FIP	2796–2814	5′-GGATGGTGTCGGAAAATTG
				+TTTT+
			2761–2779	CCTAACGGTTCGGAAGAAA-3′
		BIP	2855–2873	5′-CGTCGCGACGTGTCCCCAA
				+TTTT+
			2876–2894	CCACTCAAAGGTGTCATCA-3′
Nested RT-PCR	ORF7	N1F	14745–14766	5′-TCGTGTTGGGTGGCAGAAAAGC-3′
		N1R	15207–15228	5′-GCCATTCACCACACATTCTCCC-3′
		N2F	14867–14888	5′-CCAAATGCTGGGTAAGATCATC-3′
		N2R	15081–15102	5′-CAGTGTAACTTATCCTCCCTGA-3′

Position is marked according to the sequence of HPBEDV strain (GenBank accession number EU236259).

Fig. 1

(A) Agarose gel (2.5%) electrophoresis RT-LAMP products. Lines M, markers DL2000 (2000, 1000, 750, 500, 250 and 100 bp); lane 1, negative control; lane 2, highly pathogenic PRRSV (JXA1); lanes 3–5, highly pathogenic PRRSV blood, semen and lung samples (GD). (B) Sensitivity of nested RT-PCR determined by agarose gel electrophoresis of nested RT-PCR products (236 bp) from spiked with 5-fold dilution of the highly pathogenic PRRSV RNA (HPBEDV). Lines M, markers DL2000; lane 1, negative control; lanes 2–7, different highly pathogenic PRRSV RNA copy numbers of nested RT-PCR (1, 5, 25, 125, 625 and 3125 copies/tube, respectively). (C) Sensitivity of RT-LAMP determined by agarose gel electrophoresis of RT-LAMP products from spiked with 5-fold serial dilution of the highly pathogenic PRRSV RNA (HPBEDV). Lines M, markers DL2000; lanes 1–6, different highly pathogenic PRRSV RNA copy numbers of RT-LAMP (1, 5, 25, 125, 625 and 3125 copies/tube, respectively); lane 7, negative control. Nested RT-PCR products showed a specific amplification for the HPBEDV ORF1a with a detection limit of 25 copies, whereas detection limit of RT-LAMP is 5 copies/tube.

RT-LAMP was also compared with nested RT-PCR for direct detection in clinical specimens. Positive reactions were confirmed in all of the 122 samples by RT-LAMP and nested RT-PCR. The results indicated that this diagnostic technique was reliable for the detection of highly pathogenic PRRSV in blood, semen and lung tissue samples. Semen and blood are the preferred samples during the early stage of infection, which may have a higher predictive value of detecting highly pathogenic PRRSV infection during disease surveillance screening. Importantly, the early detection of highly pathogenic PRRSV suggests potential value as a surveillance tool in areas free of the disease and as a screening assay for monitoring an outbreak.

The test indicated that the detection limit of nested RT-PCR was 25 copies/tube (Fig. 1B) and that of RT-LAMP was 5 copies/tube (Fig. 1C). The sensitivity of RT-LAMP was therefore higher than nested RT-PCR. In addition, compared with nested RT-PCR, RT-LAMP is convenient, rapid, and sensitive. The reaction time of RT-LAMP method is 45 min, which is more rapid than conventional RT-PCR or nested PCR, and the reaction only needs a laboratory water bath. From a practical point of view, RT-LAMP is more suitable as a routine diagnostic tool, especially in clinics in which complicated equipment such as thermal cycling machines and electrophoresis apparatus are not available. In addition, RT-LAMP has a potential for field diagnosis.

In conclusion, RT-LAMP assay is rapid, specific, and sensitive for the detection of highly pathogenic PRRSV in blood, semen and lung tissue samples. This method not only reduced the diagnosis time significantly but also has a potential for wider use.