key: cord-0768458-emoexjwq
authors: Chen, Hao-tai; Zhang, Jie; Yang, Sheng-hai; Ma, Li-na; Ma, Yan-ping; Liu, Xiang-tao; Cai, Xue-peng; Zhang, Yong-guang; Liu, Yong-sheng
title: Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification
date: 2009-02-12
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2009.02.005
sha: 39a8b29aed8fba1a6ad67e78885e998277236bb8
doc_id: 768458
cord_uid: emoexjwq

A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine parvovirus (PPV) DNA. The amplification could be finished in 45 min under isothermal condition at 62 °C by employing a set of four primers targeting VP2 gene of PPV. LAMP assay showed higher sensitivity than PCR, with a detection limit of 5 copies of PPV genomic DNA per reaction. No cross reactivity was observed from the samples of other related viruses including canine parvovirus, parvovirus B19, porcine circovirus type 1, porcine circovirus type 2 and porcine peudorabies virus. The detection rate of PPV LAMP for 125 clinical samples was 97.6% and appeared higher than that of PCR method. The result indicated the potential usefulness of the technique as a simple, rapid procedure for the detection of PPV.

Porcine parvovirus (PPV) genome, a single-stranded DNA comprising about 5000 nucleotides, contains two large open reading frames (ORFs). One ORF encoding the non-structural (NS) proteins is highly conserved among the parvoviruses and the other ORF encoding capsid proteins (VP1, VP2 and VP3) may vary between some strains of PPV. VP1 and VP2 are proteins translated in the same frame but from different start codons and VP3 is a product of VP2 proteolytic cleavage (Molitor et al., 1983; Ránz et al., 1989; Bergeron et al., 1993) . PPV is considered an important cause of reproductive failure in swine. Embryonic death and resorption, mummified foetuses and stillbirth, as a result of prolonged farrowing intervals, are typical clinical signs of PPV-induced reproductive failure (Mengeling et al., 2000) . PPV occurs worldwide with variable reported prevalence rates (Maldonado et al., 2005) . The prerequisite for controlling the disease is rapid and accurate identification of this organism.

PPV isolation on cell culture is laborious and cannot be achieved for all PPV strains. Other diagnostic techniques such as ELISA (Westenbrink et al., 1989; Jenkins, 1992; Hohdatsu et al., 1998) , in situ hybridization (Waldvogel et al., 1995) and PCR have been developed (Molitor et al., 1991; Soares et al., 1999) . Although these assays are commonly accepted because of their high sensitivity and specificity, the specialized equipment and technical expertise are required.

A novel nucleic acid amplification method, termed loopmediated isothermal amplification (LAMP), was developed originally by Notomi et al. (2000) . The most significant advantages of LAMP are the ability to amplify specific DNA sequences under isothermal conditions between 63 • C and 65 • C and the result can be obtained within 60 min. The method had been applied successfully to the detection of porcine reproductive and respiratory syndrome virus, influenza A virus, severe acute respiratory syndrome coronavirus and Newcastle disease virus (Chen et al., 2008; Pham et al., 2005; Poon et al., 2005) . In this study, we evaluated the potential of LAMP for the development of a simple and rapid detection system for PPV.

The strains used in this study included PPV-BJ vaccine strain and PPV-GS, PPV-JX, PPV-SC isolated strain, PPV-GS strain was used to develop a LAMP method and determine the detection limit of the method. Monolayers of PK-15 cells grown in 15-cm 2 cul- ture flasks were infected with an inoculum of the PPV strains and the supernatant from the infected culture was collected, centrifuged at 1000 × g for 10 min and stored in aliquots at −80 • C until use. Other field isolates of canine parvovirus (CPV), parvovirus B19 (B19 V), porcine circovirus type 1 (PCV1), porcine circovirus type 2 (PCV2) and pseudorabies virus (PRV) were identified by sequencing.

A total of 125 clinical samples that were diagnosed as PPVpositive were shown in Table 1 , including blood and tissues of lymph node, lung, liver, kidney, heart, spleen, duodenum and jejunum. These clinical samples were taken from PPV-antibodypositive pigs tested by ELISA. Among them, 117 samples were identified as positive by PCR and sequencing and the other 8 samples were identified by virus isolation.

DNA was extracted from blood, lymph node, lung, liver, kidney, heart, spleen, duodenum and jejunum samples taken from PPV-infected and healthy pigs, using a DNeasy Tissue Kit (Qiagen) according to the manufacturer's instructions. After extraction, DNA was eluted in 60 l of elution buffer and stored at −20 • C.

Conventional PCR for the detection of PPV was performed with primers described previously (Arnauld et al., 1998) . Primers F, B, FIP and BIP for PPV LAMP were shown in Table 2 . A set of 4 primers for PPV LAMP were designed by targeting conserved regions of VP2 gene. VP2 nucleotide sequences of PPV strains were retrieved from the GenBank and aligned using the software program DNAStar (DNASTAR, Inc., Madison). The accession numbers used for the alignment were the following: A26477, AY390557, AY459350, AY502115, AY583318, AY597052, AY686602, AY781130, M32787, AY786299-AY786303, AY788086-AY788089, D00623, L23427, M38367, NC 001718, U44978. LAMP reaction was carried out in a conventional water bath by mixing 2.0 M each of FIP and BIP primer, 0.2 M each of F and B primer, 1.0 mM each deoxynucleoside triphosphate, 8U of Bst DNA polymerase (New England Biolabs) using the manufacturer's supplied 10× buffer (containing 2 mM of MgSO 4 , 0.8 M betaine) and 1 l of extracted template DNA in a 0.2 ml Eppendorf tube. The amplification reaction was performed at 62 • C for 45 min and then terminated by heating at 80 • C for 10 min. LAMP products were analyzed by 2.5% agarose gel electrophoresis.

The detection limit of LAMP was tested and compared with PCR by using the same templates at identical concentrations and this was done in triplicate at each concentration of templates. A recombinant plasmid pUC-19-VP2 containing 1737-bp fragment of VP2 gene of PPV-GS was constructed. Serial dilutions of 1, 5, 5 2 , 5 3 and 5 4 copies of VP2 gene per reaction from PPV strains were used in the assay. To assess the specificity of LAMP, potential cross reactions with DNA of CPV, B19V, PCV1, PCV2 and PRV were examined. Genomic DNAs of PPV-GS, PPV-BJ, PPV-SC, and PPV-JX were used as the positive control and DNA extracted from healthy swine tissues was used as the negative control. In addition, in order to evaluate the optimal tissues for viral detection and to compare the sensitivity of PPV detection between the LAMP and the PCR method, DNAs from 125 tissue samples of blood, heart, lung, liver, kidney, lymph nodes and spleen from PPV-infected pigs were extracted and subjected to LAMP and PCR.

A successful LAMP reaction produced many bands of different sizes upon agarose electrophoresis, since LAMP products consisted of several inverted-repeat structures. The amplification by LAMP showed a ladder-like pattern (Fig. 1) . The result indicated that the detection limit of the PPV LAMP was 5 copies whereas that of PCR was 25 copies per reaction (Table 3 ). The detection sensitivity of the PPV LAMP was therefore 5-fold better than that of the PCR.

DNA extracted from tissues of healthy animals, pigs infected with CPV, B19V, PCV1, PCV2 and PRV were used as templates for PPV LAMP. Agarose gel electrophoresis analysis indicated that PPV LAMP reaction did not detect CPV, B19V, PCV1, PCV2 or PRV and gave a negative reaction with tissues of healthy swine. Only with templates of PPV DNA did PPV primer set give the positive reaction. 

The general sensitivities of PPV LAMP were higher than the conventional PCR. There was a 100% positive detection rate for both PCR and LAMP on extracts of lung, kidney, heart, duodenum and jejunum tissues. However, LAMP showed higher sensitivity than PCR for the detection of PPV DNA in blood, lymph nodes, liver and spleen tissue samples. The detection rates of PCR and LAMP were 93.6% and 97.6% for the 125 clinical samples, respectively (Table 1) .

PMWS was first observed in piglets of a high-health herd in Canada in 1991 (Harding and Clark, 1997) , and appeared to be an emerging disease that affected swine herds in many countries of North America, Europe and Asia (Rodriguez et al., 1999; Rovira et al., 2002; Segalés and Domingo, 2002; Woodbine et al., 2007) . PPV was commonly found in pigs with PMWS (Woodbine et al., 2007) . Several researchers reported that PCV1, PCV2 and PRV could also reproduce symptoms typical of PMWS (Rodriguez et al., 1999; Rovira et al., 2002; Kim and Chae, 2003; Woodbine et al., 2007) . Thus, the development of a simple and rapid diagnostic tool that could detect PPV and differentiate it from PCV1, PCV2 and PRV in the same samples would be of significance for epidemiological surveillance and prediction of the severity of PMWS outbreaks in swine herds. LAMP is a new diagnostic method which is quite simple, requiring only a conventional water bath or heat block for incubation under isothermal conditions. Another useful feature of LAMP is that its products can be observed directly by naked eye, because a white precipitate of magnesium pyrophosphate forms in the reaction tube (Mori et al., 2001) . Adding SYBR Green I to LAMP reactions can increase the ease and sensitivity of detection by the naked eye (Iwamoto et al., 2003) .

The greater sensitivity of LAMP (as compared to PCR) for detecting PPV detection accords with the sensitivity reported for LAMP methods used to detect Japanese encephalitis virus, mumps virus and West Nile virus (Parida et al., 2006 (Parida et al., , 2004 Okafuji et al., 2005) . The lack of cross reaction observed with CPV, B19V, PCV1, PCV2, PRV and host-derived DNA indicates that PPV LAMP system may be specific in addition to high sensitivity. In the present study, the optimal tissues for PPV LAMP are probably the blood, lymph node, lung, kidney, heart, duodenum and jejunum because these gave a 100% detection rate in LAMP assay. Furthermore, blood is the preferable sample during the early stage of infection, which may have a higher predictive value of detecting PPV infection during disease surveillance screening.

LAMP is a simple and timesaving procedure, allowing results to be obtained within 1 h, whereas PCR method typically requires 2-4 h. Compared with PCR, LAMP method appears to be a fast and sensitive tool for the clinical diagnosis of PPV infection. Nonetheless, the reliability of this assay should be further evaluated by large-scale investigation.

Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of porcine parvovirus and application to diagnosis in piglet tissues and human plasma

Genome organization and mapping of transcription and translation products of the NADL-2 strain of porcine parvovirus

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

Recognizing and diagnosing postweaning multisystemic wasting syndrome (PMWS). Swine Health Prod

Detection of antibodies against porcine parvovirus in swine sera by enzyme-linked immunosorbent assay

Loop-mediated isothermal amplification for direct detection of mycobacterium tuberculosis complex, M. avium, and M. intracellulare in Sputum Samples

An enzyme-linked immunosorbent assay for detection of porcine parvovirus in fetal tissues

A comparison of the lymphocyte subpopulations of pigs experimentally infected with porcine circovirus 2 and/or parvovirus

Identification of viral pathogens in aborted fetuses and stillborn piglets from cases of swine reproductive failure in Spain

The effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance

Porcine parvovirus: virus purification and structural and antigenic properties of virion polypeptides

Polymerase chain reaction (PCR) amplification for the detection of porcine parvovirus

Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation

Loop-mediated isothermal amplification of DNA

Rapid diagnostic method for detection of mumps virus genome by loopmediated isothermal amplification

Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus

Development and evaluation of reverse transcription-loop-mediated isothermal amplification assay for rapid and realtime detection of Japanese Encephalitis virus

Loop-mediated isothermal amplification for rapid detection of Newcastle disease virus

Detection of human influenza A viruses by loop-mediated isothermal amplification

Porcine parvovirus: DNA sequence and genome organization

Aujeszky's disease virus infection concurrent with postweaning multisystemic wasting syndrome in pigs

Experimental inoculation of conventional pigs with porcine reproductive and respiratory syndrome virus and porcine circovirus 2

Postweaning multisystemic wasting syndrome (PMWS) in pigs. A review

Detection of porcine parvovirus DNA by the polymerase chain reaction assay using primers to the highly conserved nonstructural protein gene, NS-1

Diagnosis of fetal infection with porcine parvovirus by in situ hybridization

An enzyme-linked immunosorbent assay for detection of antibodies to porcine parvovirus

Spatiotemporal patterns and risks of herd breakdowns in pigs with postweaning multisystemic wasting syndrome

This work was supported in part by grants from the National Key Technologies R&D Program of China (Nos. 2006BAD06A03 and 2008BADB4B05). This study was also supported by the National Institutes R&D Program of China (BRF080305) and the Natural Science Foundation of Gansu Province, China (No. 0803RJZA050).