Establishment of Real-Time TaqMan-Fluorescence Quantitative RT-PCR Assay for Detection and Quantification of Porcine Lipoprotein Lipase mRNA

Porcine lipoprotein lipase （LPL） cDNA was cloned as the standard for real-time quantifying LPL mRNA and the TaqMan-fluorescence quantitative PCR assay for detection was established. The total RNA extracted from Longissimus dorsi of porcine was reverse-transcribed to cDNA. LPL cDNA was ligated with pGM-T vector and transformed into Escherichia coli TOP10. Plasmid DNA extracted from positive clones was verified by PCR amplification and sequenced. LPL was amplified by real-time fluorescence quantitative PCR from the plasmid DNA. The concentration of DNA template purified was detected by analyzing absorbance in 260 nm and then the combined plasmid was diluted to series as standard for fluorescence quantitative PCR （FQ-PCR）. The method of LPL mRNA real-time PCR was well established, which detected as low as 103 with the linear range 10^3 to 10^10 copies. The standard curves showed high correlations （R2 = 0.9871）. A series of standards for real-time PCR analysis have been constructed successfially, and real-time TaqMan-fluorescence quantitative RT-PCR is reliable to quantitatively evaluate FQ-PCR mRNA in L. dorsi of porcine.

Taqman PCR-based Methods for Rapid Detection of Salmonella in Pet Food

[ Objective] To establish a Taqman real-time PCR for detection of Salmonella in pet food. [Method] A pair of primers and a probe were designed based on published nucleotide sequence of invA gene encoding the invasion protein of Salmonella enterica. [ Result] The assay detects Salmonella specifically. The detection limit of the real-time PCR was 17 CFU/test （25 uL/test） for the positive strain. This method was effective to detect artificially contaminated pet food. [ Conclusion] The results showed that Taqman PCR assay was rapid and accurate for detection of Salmonella from infected pet food.

Establishment of a Real-time Fluorescent Quantita- tive RT-PCR Method for Pineapple mealybug wilt as. sociated virus-2

Pineapple mealybug wilt associated virus-2 （PMWaV-2） is an important pathogen causing Mealybug wilt of pineapple （MWP）. We established a realtime quantitative RT-PCR （RT-qPCR） method with TaqMan probe based on specific primers of conserved nucleotide sequence of PMWaV-2 coat protein gene. The results showed that the method was higldy sensitive to positive sample, but had no fluorescence signal to health sample and blank control. The sensitivity of RTqPCR was about 100 times higher than general PCR. Reproducibihty test revealed that the coefficients of variation between intra-and inter-assay were beth within 1.85%, indicating it was a quantitative detection method for PMWaV-2 with simple operation, strong specificity, high sensitivity, and reliable reproducibility.

Development of Quantitative Real-time Polymerase Chain Reaction for the Detection of Vibrio vulnificus Based on Hemolysin (vvhA) Coding System

Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene （vvhA） coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles （Ct value） and fluorescence intensity enhancement （ARn value） were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.

SNP Genotyping of Myostatin (MSTN) Gene through Taq Man Probe Assay and Association Study between MSTN Genotypes and Growth Traits of Tan Sheep

To establish a rapid, accurate and economical real-time PCR assay system based on TaqMan probe technology for the detection of genetic variations of single nucleotide polymorphisms （SNPs） in myestatin （MSTN） gene, a pair of TaqMan probes were designed on the polymorphism loci of MSTN gene and used in PCR reaction system for SNP genotyping. Meanwhile, an association study was performed between MSTN genotypes and growth traits of Tan sheep, including birth weight, weaning weight, 3-month weight, and 6-month weight. The results showed that rs417816017 locus of MSTN gene in Tan sheep had two genotypes ： YY and XY. The individuals with genotype XY had a growth advantage over the ones with genotype YY. The results indicate that TaqMan probe-based real-time PCR assay can be used to detect the genotype of MSTN gene, which will provide candidate genes for breeding of Tan sheep.

Establishment of Real-time Fluorescent Quantitative PCR for Pineapple mealybug wilt associated virus-3

[ Objective ] The paper aimed to establish a real-time fluorescent quantitative PCR （qPCR） detection method for Pineapple mealybug wilt associated vi- rus-3 （ PMWaV3 ）. [ Method] Specific TaqMan probe and primers were designed and synthesized according to the conserved sequence of coat protein（CP） gene of PMWaV-3, and the standard curve was established after optimizing the amplification condition of qPCR. [ Result] The results showed that the method was specific for the detection of PMWaV-3, and the sensitivity of the present method was about 10 times higher compared to general RT-PCR ; the variation coefficients of intra- assay and inter-assay were less than 1.73, respectively. [ Conclusion] The qPCR is an easy, fast and reliable method for quantitative detection of PMWaV-3.