Sensitive and rapid detection of Mycoplasma capricolum subsp . capripneumoniae by Loop-mediated isothermal amplification

A set of four specific primers was designed by targeting the H2 gene sequences of Mycoplasma capricolum subsp. capripneumoniae (MCCP). Using Bst DNA polymerase, the products were amplified for 60 min at 65°C in a simple water bath. Compared with a polymerase chain reaction (PCR) test that targets the H2 gene sequences of MCCP, the sensitivity of the loop-mediated isothermal amplification (LAMP) assay was higher (approximately 0.75 fg DNA per reaction). The LAMP products could be visualized by agar gel electrophoresis. There were no cross reactions with other strains in the Mycoplasma mycoides cluster, which indicates the high specificity of the LAMP procedure. The LAMP assay was able to detect MCCP in tissue.

CCPP is classified as a list B disease by the Office International Des Epizooties or World Organization for Animal Health (OIE).It is an infectious disease that *Corresponding author.E-mail: luzhongxin@hotmail.com;Tel: 86 931 8342676.
Author(s) agree that this article remain permanently open access under the terms of the Creative Commons Attribution License 4.0 International License Abbreviations: CCPP, Contagious caprine pleuropneumonia; MCCP, Mycoplasma capricolum subsp.capripneumoniae; LAMP, loop-mediated isothermal amplification; PCR, polymerase chain reaction.affects only goats, and was first described in the late 19th century (Hutcheon, 1889;McMartin et al., 1980).Before the isolation and identification of Mycoplasma strain F38 by MacOwan (1976) and the subsequent demonstration of its causal relationship with CCPP (MacOwan and Minette, 1976), M. mycoides subsp.capri was considered to be the aetiological agent of CCPP (Edward, 1953;JonAs and Barber, 1969).So far, M. capricolum subsp.capripneumoniae is the only mycoplasma that fulfills the Koch postulates for CCPP, and it is believed to be the sole cause of CCPP (MacOwan, 1984).Mycoplasma strain F38 has been reclassified recently, and now all F38-like mycoplasmas are known as Mycoplasma capricolum subsp.Capripneumoniae (Leach et al., 1993).
CCPP has been reported to affect only goats (Thiaucourt and Bolske, 1996) and it does not cause disease in sheep, either spontaneously or experimentally (McMartin et al., 1980).However, there are some reports that describe the isolation of M. capricolum subsp.capripneumoniae from healthy sheep in Kenya that had been in contact with goat herds affected by CCPP (Litamoi et al., 1990), and from sick sheep in Uganda that had been mixed with goats suffering from the disease (Bolske et al., 1995).The isolation of M. capricolum subsp.capripneumoniae from cattle with mastitis has also been reported (Kumar and Garg, 1991), and these reports contradict the perceived host specificity of M. capricolum subsp.capripneumoniae.
Recently, a novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), has been reported (MacOwan and Minette, 1976).The LAMP assay is rapid and its amplification efficiency is equivalent to that of polymerase chain reaction (PCR)based methods (Cai et al., 2010;Gadkar and Rillig, 2008).More importantly, the approach is less costly, and all reactions can be developed in an isothermal environment.Reports of the detection of MCCP using LAMP assays have shown that the approach is easier and faster to perform than conventional PCR assays, as well as being more specific (Endo et al., 2004).In this study, a method based on the LAMP assay for the detection of CCPP was developed, and the sensitivity and specificity of the assay were evaluated.The assay was compared with a PCR test targeting the H2 gene sequences of MCCP.

Strains and cultivation
The origin of the 21 stains used in this study, type strains of the M. mycoides cluster and field isolates from China, are listed in Table 1.The mycoplasmas were cultivated in modified KM 2 (Hanks solution with 1.7% lactalbumin hydrolysate, 1% MEM, 20% decomplemented horse serum, 5% fresh yeast extract, 1% thallium acetate, 0.4% sodium pyruvate) in a high security laboratory.The DNA of Pasteurella multocida and Mannheimia haemolytica was maintained at the State Key Laboratory of Veterinary Etiological Biology.

Clinical samples
Twenty-eight (28) samples from 14 goats infected artificially with M. capricolum subsp.capripneumoniae were used in the study.The clinical samples were collected when the goats showed primary clinical signs: Cough, anorexia, laboured breathing with painful grunting, and a rise in temperature up to 41°C.Given that the gross pathological lesions were localized exclusively in the lung, the livers were collected for use in the experiment (Table 1).Sixty-one (61) clinical samples collected from western China in 2009-2011 were used for the epidemiological survey.These samples were kept at -80°C until analysis.

Sample preparation
Culture samples (1 ml) were centrifuged at 1,2000 RCF for 20 min at 4°C.The cell debris was pelleted; the pellets were washed in phosphate buffered saline (PBS) and re-suspended in 50 μl ddH 2 O.After vortexing, the samples were lysed by boiling for 10 min, centrifuged and diluted 1:50.For the clinical samples, DNA extraction was performed using a kit (DNA extraction kit, Invitrogen, Carlsbad, USA) according to the protocol of the manufacturer.The extracted DNA was used for PCR both undiluted and at 1:50 dilution.

LAMP primers
The MCCP LAMP primer set was designed using the primerExplorer program http//primerexplorer.jp/e/index.htmlto amplify the H2 gene (GenBank access number: AF162991.1).The primers are shown in Table 2.

LAMP method
The LAMP reactions were carried out with a 25 μL reaction mixture containing 2 μL of extracted DNA, 40 pmol (each) of primers FIP and BIP, 5 pmol (each) of primers F3 and B3, 2.8 mM of each dNTP, 4 U of the large fragment of Bst DNA polymerase (Bst DNA polymerase (Biolabs @ inc., New England, USA)), with the corresponding polymerase buffer.The reaction temperature and time were 65°C and 60 min.The reaction was terminated by heating at 80°C for 3 min.Positive and negative controls were

Specificity of LAMP
To determine the specificity of the LAMP method, it was carried out at 65°C for 60 min with the DNA of various mycoplasmas in the M. mycoides cluster; the templates from the eight type strains of the M. mycoides cluster and Pasteurella multocida are listed in Table 3.Each DNA sample from the strains tested was examined in triplicate.The products were separated by 2% agarose gel electrophoresis, and the target bands were visualized by staining with ethidium bromide.

Sensitivity of the LAMP method
The sensitivity of the assay was assessed by testing tenfold serial dilutions of 1 μg ml -1 DNA of M. capricolum subsp.capripneumoniae F1601.Reaction mix without the DNA template was included as a negative reaction control.The LAMP amplification products were analyzed visually by 2% agarose gel electrophoresis.To compare  the detection sensitivities of LAMP and PCR, PCR using H2 gene primers that amplify a 680-bp product was carried out in a total reaction volume of 25 μL containing 2 μL of the MCCP DNA, 2 μL (10 pmol ml -1 ) of a pair of appropriate primers, 12.5 μL Premix Ex Taq (TaKaRa, Shiga, Japan) and ddH 2 O.The PCR conditions consisted of an initial denaturation step of 5 min at 94°C, followed by 35 cycles of 45 s at 94°C, 30 s at 50°C and 45 s at 72°C, and a final extension step of 10 min at 72°C.Samples of the PCR amplification products (5 μl) were analyzed by 1% agarose gel electrophoresis.Tests of the sensitivity of the LAMP and PCR assays were conducted in triplicate, and the detection limits were defined as the last positive dilutions, with a sample being considered positive if all three samples tested positive.

Specificity of the LAMP assay
The specificity of LAMP was tested using DNA extracted from the eight type strains of the M. mycoides cluster and P. multocida.After incubation at 65°C for 60 min, MCCP was positively detected, whereas no other mycoplasma isolate was detected or amplified by LAMP.The other type strains of the M. mycoides cluster and P. multocida, as listed in Table 3, were negative (Figure 1).

Sensitivity of the LAMP assay
To assess the sensitivity of the LAMP assay for the detection of MCCP, the reaction was tested using 1 μL tenfold serial dilutions of MCCP DNA and compared with the PCR assay.The LAMP reaction was able to detect up to 0.75 fg DNA per reaction (Figure 2); however, the PCR could only detect MCCP up to 750 pg per reaction (Figure 3).The results indicate that LAMP has a higher sensitivity than the standard PCR method.

Clinical samples
Twenty-eight (28) clinical samples originating from 14 animals showing serious clinical signs were included in the analysis: 28 samples were positive by LAMP assay, using  1:50 diluted templates (Figure 4).To evaluate the LAMP test further, comparison with the PCR test was performed using samples from an epidemiological survey conducted in western China.The results are shown in Table 4. Overall, from 61 clinical samples, the LAMP assay gave a total of 11 more positive results than the PCR test.

DISCUSSION
The LAMP primers used in this study were based on the H2 gene sequence, which confers some advantages for molecular identification.The H2 gene is a putative membrane protein gene.It is reported that the partial sequence of the H2 gene can be used as an epidemiological marker for the M. mycoides cluster because it is a conserved sequence.Such studies demonstrate the use of the H2 gene for the molecular identification of closely related genomic species (Thiaucourt and Bolske, 1996).LAMP method compared with conventional PCR reported in this article has the advantages of simple operation, rapid reaction and ease of detection.The LAMP assay is a simple detection tool in which the reaction is performed in a single tube by mixing the thermopol buffer, primers and Bst DNA polymerase, followed by incubation of the mixture at 65°C for 60 min.The LAMP reaction is performed under isothermal conditions and it does not require expensive equipment: the only equipment needed for the LAMP reaction is a regular laboratory water bath or a heating block that can provide a constant temperature of 65°C.Moreover, the amplification efficiency is extremely high, there is no time required for thermal cycling, and inhibition reactions at later stages are less likely to occur than in standard PCR.In addition, LAMP amplifies DNA to higher concentrations than PCR, allowing convenient visualization of the products after the addition of SYBR Green I without gel electrophoresis.Hence, the LAMP assay could be developed into a field test.
In this study, the LAMP method for the detection of MCCP was found to be highly sensitive, because it could detect MCCP at 0.75 fg DNA per reaction, whereas by PCR, the detection of MCCP was possible only up to750 pg DNA per reaction.This indicates that the sensitivity of LAMP is higher than that of the standard PCR.The increased sensitivity may make LAMP a better choice than PCR for the detection of MCCP from cases of in apparent infection.
In conclusion, the LAMP method described in this study represents a new, sensitive, specific, and rapid protocol for the detection of MCCP.It may be applied in epidemiological surveys of contagious caprine pleuropneumonia.

Table 1 .
Isolation of Mycoplasma capricolum subsp.capripneumoniaeand LAMP and PCR-detection from artificial infected animals with MCCP.

Table 2 .
Primer sequences used in this study.

Table 3 .
Collection of strains used to test the specificity of the Mycoplasma capricolum subsp.capripneumoniaeLAMP.

Table 4 .
Isolation of Mycoplasma capricolum subsp.capripneumoniaeand LAMP and PCR-detection from clinical samples obtained from animals suspected for being infected with MCCP.