Development of a loop-mediated isothermal amplification method to rapidly detect porcine circovirus genotypes 2a and 2b

Background Porcine circovirus type 2 (PCV2), is nowadays associated with a number of diseases known as porcine circovirus-associated diseases (PCVAD), especially postweaning multisystemic wasting syndrome (PMWS). The epidemiological investigation of PCV2 infection was usually conducted by PCR, nested PCR, PCR-RFLP, TaqMan-based assay and nucleotide sequencing. However, there is still no rapid, sensitive and practical method for detecting PCV2 genotypes. As a novel nucleic acid amplification method, the loop-mediated isothermal amplification method (LAMP) has been used to detect a variety of pathogenic microorganisms. Results Herein, a LAMP method is developed to detect the genotypes of PCV2. The diagnostic sensitivity of LAMP is 1 copy/reaction for differentiating genotypes PCV2a and PCV2b. The reaction process was completed at 65°C for 1 hour in a water bath. Cross-reactivity assay shows that this method is specific for PCV2a and PCV2b and no reactive for PCV2c and other swine-origin viruses (i.e. CSFV, PRRSV, BVDV, TGEV and PEDV, etc). Identity between LAMP and nested PCR was 92.3% on 52 field clinical samples. Conclusions LAMP method provides a rapid, sensitive, reliable way to detect PCV2a and PCV2b, and a better means for the large scale investigation of PCV2a and PCV2b infection.

LAMP method with high DNA specificity was developed in 2000 as a novel nucleic acid amplification method [22]. The use of the Bst DNA polymerase with high strand displacement activity leads to a few copies of DNA can be amplified to 10 9 in less than an hour under isothermal conditions [22]. Therefore, LAMP is simple and easy to perform once the appropriate primers are prepared. Thus far, LAMP method has been used to detect a variety of pathogenic microorganisms [23][24][25], including PCV2 [21,26]. However, the LAMP method used to detect PCV2 genotypes has not yet been reported. In this study, a LAMP assay was developed to provide a rapid, sensitive, reliable method for PCV2 genotypes detection.

Optimized LAMP assay for PCV2a and PCV2b
The concentrations of the forward inner primers (FIP) and backward inner primers (BIP), deoxynucleoside triphosphates (dNTPs), betaine and the reaction temperature were optimized when the LAMP assay was developed. The optimal LAMP reaction mixture (25 μL) was composed of 0.8 μM (each) of inner primers, 0.2 μM (each) of outer primers, 0.2 mM of dNTPs, 0.5 M of betaine, 10 × ThermoPolbuffer 2.5 μL, 8U of Bst DNA polymerase, and 1 μL of template DNA. The mixture was incubated at 65°C for 1 hour, inactivated at 80°C for 10 min, and characterised a bolder ladder like bands on 2% (W/V) agarose gel under UV light after EB staining (data not shown). The similar results can also be observed by naked eyes after adding SYBR Green I dye (data not shown). Figure 1A showed that PCV2a and PCV2b could be detected by their special LAMP primers respectively, the same templates were amplified by nested PCR as positive control ( Figure 1A, Lane 1). But the genotype PCV2c could not be detected (data not shown). Under the identical condition, none of PCV1, CSFV, PRRSV, BVDV, TGEV, PEDV and RV could give a positive reaction with the two sets of LAMP primers ( Figure 1B, Lanes 2, 3), while the same templates were amplified by conventional PCR with their corresponding special primers respectively ( Figure 1B, Lane 1). These data demonstrated that the LAMP assay was specific for PCV2a and PCV2b.

Data in
Sensitivity of the LAMP assay and nested PCR assay Sensitivity testing revealed the detection limit of the LAMP assay is 1 copy/reaction for both of PCV2a and PCV2b (Figure 2A), while the detection limit of the nested PCR was 10 3 copies/reaction for both of PCV2a and PCV2b ( Figure 2B), indicating the sensitivity of the LAMP method was elevated one thousand times in comparison with the nested PCR.

Stability of the LAMP assay
Of four positive templates of PCV2a and PCV2b, three replications of each template appeared the same pattern and brightness of the amplified bands. Although the brightness of the amplified bands varied according to the different concentration of the templates, the pattern of the amplified bands is consistent. These results indicated the LAMP method was stable ( Figure 3).

PCV2a and PCV2b detection of clinical samples
Of the 52 clinical samples, 52 samples were positive for PCV2b and 4 samples were positive for genotypes PCV2a in the LAMP method, indicating that PCV2b is the major epidemic genotype and co-infection of PCV2a and PCV2b in pig farms. In the nested PCR amplification, 48 of 52 samples were positive for PCV2b and no PCV2a was detected. These data showed that the identity between LAMP and nested PCR was 92.3% (48/52) and the LAMP method had higher sensitivity than the nested PCR method.

Discussion
Generally, the nucleotide sequencing has been used to detect PCV2a and PCV2b [12], other approaches included PCR [16], nested PCR [17], PCR-RFLP [18] and TaqMan-based assay [27]. However, these methods were usually time consuming and high technical requirement, and were difficult to popularize in veterinary clinic practice. In the present study, the LAMP method was developed for detecting PCV2 genotypes, and it was a simple, rapid and easy operation for veterinarians.
The specific genotype primers for differential PCR amplification with high sensitivity and high specificity are not easy to design, because the genetic identity between PCV2a and PCV2b is very high (>95%) [28]. However, the nucleotides sites 1480-1469 of PCV2a and PCV2b are ACCAACAAAAT and TCAAACCCCCG(T), respectively [12], therefore, we can distinguish PCV2a and PCV2b according to this tag motifs. In this study, the special LAMP primers were designed respectively based on the tag motifs of PCV2a's AT rich area and PCV2b's GC rich area of the ORF2 gene, and the homology of all these primer sequences among 372 PCV2 strains is over 90%, indicating that these primers could deal with most of the PCV2 strains in LAMP assay. During the development of the LAMP  assay, the concentration of outer primers is fixed, and 0.4 μM, 0.6 μM, 0.8 μM is selected for optimizing the concentration of inner primers, because the outer and inner primers are used in the initial steps of the LAMP reaction and the inner primers are used for strand displacement DNA synthesis at late stage of nucleic acid amplification [22]. It has been reported that the addition of 0.5-1.6 M betaine improved the amplification of DNA by reducing the formation of secondary structure in GC-rich regions [29], and 0.5 M, 1 M, 1.5 M was selected for the concentration of betaine optimization. In addition, we found that the efficiency of nucleic acid amplification falled as the concentration of dNTPs increased, and even no products. This may be attributed to that the excessive dNTPs resulted in chelation of magnesium ions in the reaction buffer.

Conclusions
Take together, we can find that the LAMP method has better specificity, higher sensitivity and efficiency, and simpler equipment requirements than the nested PCR. Furthermore, the detection of the amplification product is more flexible. Therefore, the LAMP method developed in this study provides a better means for the molecular epidemiological investigation of PCV2 genotypes.

Clinical specimens
Clinical specimens for inspection, including inguinal lymph nodes, mesenteric lymph nodes, livers and lungs, were collected from different pig farms in Zhejiang Province and its neighbour areas under the condition of the farmer's consents. All tissue samples were homogenized with a mortar in minimal essential medium at the ratio of 1:10. Then the suspension was centrifuged at 6,000 rpm for 10 min to obtain the supernatant. As a positive control, PK-15 cells infected respectively with PCV1 or PCV2a or PCV2b were frozen and thawed three times, then centrifuged at 6,000 rpm for 10 min to obtain cell free supernatant. All samples were stored at -80°C until used.

DNA and RNA extraction
The viral DNA was extracted by using the UNIQ-10 viral DNA extraction kit according to manufacturer's instructions (Sangon Biotech Co., Ltd., Shanghai, China). RNA isolation of CSFV, PRRSV, BVDV, TGEV, PEDV and RV was carried out using Trizol Reagent (Invitrogen, USA) according to manufacturer's specification. The extracted RNA was used as template after reverse transcription with random primer according to the instructions of the RevertAid TM First Strand cDNA Synthesis Kit (Fermentas, Canada). All templates were stored at -20°C until used.

LAMP primers design
According to the reference sequences of PCV2a (GenBank: AF055382) and PCV2b (GenBank:AF055394) [10], two sets of primers special for PCV2a (inner primers FIPa and BIPa, outer primers F3a and B3a) and PCV2b (inner primers FIPb and BIPb, outer primers F3b and B3b) ( Table 1) are designed based on the tag motif [12] in PCV2a's AT rich area and PCV2b's GC rich area of the ORF2 gene online (http://primerexplorer.jp/eLAMP3.0.0/index. html). Inner primers contain sequences of the sense and antisense strands of the target DNA linked by a TTTT spacer (Hightlighted in bolder pattern in Table 1). The homology of each primer among 372 PCV2 strains is calculated by MEGA (VERSION:5.0) [30]. Primers are synthesized by Sangon (Sangon Biotech Co., Ltd, Shanghai, China).

Nested PCR
The nested PCR method for detecting PCV2a and PCV2b was developed by Lyoo in 2008 [17], and it was adjusted and used to compare with the LAMP method on sensitivity. The primer synthesis and the PCR procedure were followed as described in the paper [17]. The polymerase, dNTPs, PCR buffer were replaced by the 2 × Taq PCR MasterMix (TIANGEN Biotech Co., Ltd, Beijing, China).

Specificity and sensitivity
To detect the specificity of the LAMP method, PCV2a, PCV2b, PCV1, CSFV, PRRSV, BVDV, TGEV, PEDV and RV templates were amplified with LAMP primers for PCV2a and PCV2b respectively. As compared, all these templates were detected by conventional PCR with their special primers to ensure the template for individual virus is testable.
To detect the sensitivity of the LAMP method, the concentration of the extracted DNA of PCV2a and PCV2b was quantified with ND-1000 spectrophotometer (NanoDrop, Wilmington, USA), and serial ten-fold dilution method was used to dilute it from 2.5 × 10 7 copies/μL to 2.5 × 10 1 copies/μL.

Stability
Four PCV2a and PCV2b postive templates were chosen to test the stability of the LAMP method, respectively. Each template was repeated three times under identical conditions. The amplified products (5 μL) were analyzed by agarose gel electrophoresis.