腐皮镰刀菌SYBR Green实时荧光定量PCR快速检测方法的建立

目的建立一种能够快速、灵敏、特异的鉴定腐皮镰刀菌的SYBR Green实时荧光定量PCR。方法运用SYBR Green实时荧光定量PCR反应体系检测腐皮镰刀菌,并对此方法的特异性、灵敏度和稳定性进行评价。结果通过对45例样品的检测,结果显示SYBR Green实时荧光定量PCR特异性好,其检出率高于普通PCR;灵敏度高,对重组质粒标准品的检测灵敏度为1.0×10~2copies/μL;稳定性好,对质粒为1.0×10~7copies/μL、1.0×10~5copies/μL、1.0×10~3copies/μL的标准品重复检测10次,结果显示扩增反应Ct值的变异系数为0.96%~1.68%。结论SYBR Green实时荧光定量PCR检测腐皮镰刀菌,不仅特异性好,灵敏度高,稳定性好,而且简便、快速、易操作。     

Sex determination in 6 bovid species by duplex PCR

Sex determination in domestic animals is of potential value to livestock breeding programs. The aim of this study was to develop a simple and accurate PCR-based sex determination protocol, which can be applicable to 6 major domesticated species of the family Bovidae,viz. Bos frontalis, B. grunniens, B. indicus, Bubalus bubalis, Capra hircus, andOvis aries. In silico analysis was done to identify conserved DNA sequence in the HMG box region of the sex-determining region of the Y-chromosome (SRY gene) across the bovids. Duplex PCR assay, including theSRY gene and theGAPDH housekeeping gene, was optimized by using genomic DNA extracted from blood samples of known sex. It was possible to identify the sex of animals by amplifying both gender-specific (SRY) and autosomal (GAPDH) genes simultaneously in the duplex reaction, with the male yielding two bands and the female one band. The protocol was subjected to a blind test that showed a 100 percent specificity and accuracy, thus it can be used in sex determination in livestock breeding programs.     

Plasmodium vivax: parasitemia determination by real-time quantitative PCR in Aotus monkeys

Plasmodium vivax and Plasmodium falciparum are the two prevalent human malaria species. A Colombian P. vivax wild strain has been adapted in Aotus nancymaae monkeys for use in further biological and immunological studies. We present data validating a real-time PCR assay quantifying P. vivax parasitemia, using the small subunit ribosomal RNA genes as an amplification target. P. vivax species-specific primers were designed on the 18S ribosomal gene V8 region, for amplifying both asexual and sporozoite ssrRNA genes. The assay detects amplification products bound to fluorescent SYBR-Green I dye using Perkin-Elmer GeneAmp-5700-SDS. Linear range standard curves from 6 DNA concentration logs (+0.99 correlation coefficients) were obtained. Standard curves were constructed using a plasmid containing target gene for real-time PCR amplification. This P. vivax specific assay is very sensitive, having a three parasite detection limit, and is reproducible and accurate. It involves a "closed-tube" PCR, avoids time-consuming post-PCR manipulation, and decreases potential PCR contamination.     

Sex determination and milk protein genotyping of preimplantation stage bovine embryos using multiplex PCR

A method for determining the sex and milk protein genotypes (RFLPs) of preimplantation stage bovine embryos using multiplex polymerase chain reaction (PCR) is described. Day 6 to 7 embryos were micromanipulated to isolate 5 to 6 cells. These cells were then dried in reaction tubes for transport to the laboratory. Subsequently, two sets of PCRs were performed using Y chromosome, k-casein and beta-lactoglobulin gene specific primers, followed by electrophoretic analysis of the PCR products. The presence or absence of the Y chromosome was ascertained in 90 of 92 embryos. Moreover, the k-casein specific fragment was amplified and detected in all these embryos. The PCR products were digested in order to genotype the k-casein gene. In 70% of the embryos, the beta-lactoglobulin specific fragment was amplified, although together with some unspecific fragments.     

Sex determination of bovine embryo blastomeres by fluorogenic probes

One of the major challenges of using genetic information in marker assisted selection (MAS) is the detection of multiple marker loci from a small biopsy sample of a preimplantation stage embryo. The objective of this study was to develop a fast, nested, multiplex preamplification, polymerase chain reaction (PCR) method for the determination of sex in bovine embryo blastomeres. For this aim, ZFX/ZFY sequences were preamplified simultaneously with other genomic regions. The preamplification product was used as a template in an allelic discrimination assay, with nested primers and sex specific fluorogenic probes for ZFX and ZFY. Fluorogenic probes were used to eliminate the need for time consuming electrophoresis. Compared to sexing with Bovy/kappa-casein co-amplification method and other replicates from the same embryo, the accuracy of sexing with the use of fluorogenic probes after preamplification was 99% (112/113 blastomeres). The amplification efficiency was 96% (113/117 blastomeres).     

Sensitive detection of Mycobacterium avium subsp. paratuberculosis in bovine semen by real-time PCR

AIMS: To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity. METHODS AND RESULTS: Processed semen was spiked with known amounts of MAP. Semen from different bulls as well as semen of different dilutions was tested. The samples were treated with lysing agents and beadbeating and the DNA was extracted with phenol and chloroform. Real-time PCR with a fluorescent probe targeting the insertion element IS900 detected as few as 10 organisms per sample of 100 mul semen. PCR-inhibition was monitored by inclusion of an internal control. Pre-treatment with immunomagnetic separation was also evaluated, but was not shown to improve the overall sensitivity. CONCLUSIONS: Real-time PCR is a sensitive method for detection of MAP in bovine semen. Lysis by mechanical disruption followed by phenol and chloroform extraction efficiently isolated DNA and removed PCR-inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The high sensitivity of the applied method allows reliable testing of bovine semen used for artificial insemination to prevent the spread of Johne's disease, caused by MAP.     

Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a “dilution bias” when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.     

蜜蜂性别决定与性比调控机理研究

叙述了 4个主要蜜蜂性别决定机理的假说 :即性位点假说、基因平衡假说、蜜蜂性别决定综合假说和性基因数量决定假说。然后就蜜蜂性比由蜂王操纵 ,或是由工蜂操纵进行了论述 ,并对蜜蜂性比调控机理研究提出了一些建议