Equine Reproductive Procedures. Группа авторов

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СКАЧАТЬ nucleotides. Commercial kits containing DNA polymerase and nucleotides are available to maximize the efficiency of the replication process.

       The PCR tube containing the DNA of interest, PCR primers, DNA polymerase, nucleotides, and SYBR green (fluorescent dye) are placed into a thermal cycler or transferred into a 96‐well plate with other samples and loaded into a thermal cycler that will heat and cool the tube at specific times while monitoring the amplification of DNA within the tube.

       The initial step within the thermal cycler is to heat the tube to approximately 95°C, which will separate the DNA double helix into single‐stranded DNA.

       The tube is then cooled down to approximately 50°C (122°F), which will allow the primers to lock or anneal onto their target sequence, if it is present in the PCR tube after extraction.

       Subsequently, the tube is heated to approximately 72°C (162°F) for one to several minutes, which will activate the DNA polymerase to synthesize a copy of the DNA strand template. DNA polymerase binds to the primers and only adds nucleotides in a 5´ to 3´ progression. The length of time for this step is directly dependent on the length of the gene of interest, for example it will take longer to replicate 450 base pairs of DNA than 100 base pairs. As the DNA polymerase is replicating the area of interest, a fluorescent signal is released that can be detected by the thermocycler. A stronger signal indicates that there are more copies of DNA being replicated at that specific qPCR replication cycle (Ct cycle). The fluorescent signaling provides a quantitative estimate of the amount of DNA in the sample.

       The process is then repeated over multiple cycles and an amplification curve is generated (Figure 16.1). It is estimated that 40 cycles will produce over 1 billion copies of the targeted DNA sequence from one original DNA sequence. As noted above, copies will only be produced if the targeted DNA sequence was originally present in the initial tube after extraction. The entire time period for amplification is approximately 1–2 hours.Figure 16.1 Example of an amplification curve detecting fungal DNA in a clinical sample. The y axis is the relative amount of fluorescence detected and the x axis is the number of replication cycles (Ct). There is one clinical sample (third line from the left) and seven control samples being evaluated in this particular amplification curve. The samples are going through an exponential phase of DNA replication and a corresponding amount of fluorescent signal is produced. The cycle in which the fluorescent signal surpasses the threshold is considered the Ct value in which the sample was positive. The exponential phase continues for 4–6 replication cycles before entering a plateau or non‐exponential phase. The clinical sample had a Ct value of 17 which is near the control sample at Ct cycle 16 corresponding to approximately 1,000 CFU of Candida albicans. The amplicon was analyzed for genus and species identification and the organism was determined to be Candida krusei.

       The number of qPCR cycles or Ct cycles required to produce approximately 1 billion copies of the targeted DNA sequence can be used to estimate the relative concentration of microbial DNA in the original sample, providing for a semiquantitative evaluation.

       All qPCR assays should have controls with known colony‐forming unit (CFU) standards to help control for day to day variation in extraction and replication of DNA. Additional controls of DNA‐free water should be run in tandem with the unknown sample to determine if contamination has occurred during the extraction or amplification process. DNA‐free water may exhibit evidence of contamination after approximately 35–37 Ct cycles. This key control is important to prevent a false‐positive report to the clinician that submitted the sample.

       The end product of the qPCR replication process is called an amplicon.

       The amplicon consists of millions of copies of a DNA sequence. The DNA sequence of the amplicon can be submitted for DNA sequencing and compared against known published DNA sequences of microbial organisms using the web‐based BLAST (Basic Local Alignment Search Tool) sequence‐similarity tool.

       A genetic match of DNA sequences between the DNA replicated by qPCR and a published DNA sequence identifies the genus and species of the microbial organism recovered from the uterine sample.

Interpretation of qPCR Data

The number of threshold cycles (Ct cyles) required to replicate the original microbial DNA in the extracted sample (the template DNA) above the threshold level of detection (Figure 16.1) can be used to provide an approximation of the original DNA load. This can be used to provide an approximation of the number of microbial organisms in the original sample and the degree or severity of the infection (Table 16.1). Interpretation can be confounded by many factors including extraction efficiency, qPCR sensitivity, contamination during sample collection, handling, storage, and processing, type of microbial organism, mare health status, and other factors.

Table 16.1 Interpretation of qPCR results relative to degree of infection in equine uterine samples based on studies performed at Colorado State University.

Number of Ct Cycles	Degree of Infection	Comments
≥35	None evident	Negative sample. If microbial DNA is detected, it is most likely due to contamination. Results need to be compared to negative controls
30–34	Mild	Low amount of bacterial DNA. Interpretation should include results of other diagnostic tests
20–30	Moderate	Presence of microbial DNA at a level consistent with infection
<20	Severe	A large amount of microbial DNA detected

      The lower limit of detection of the bacterial organism Streptococcus equi subspecies zooepidemicus in equine uterine fluid has been reported to be 429 × 10^–12 g, which is approximately 40 organisms worth of DNA. The lower limit of detection of DNA for the fungal organism Candida albicans has been reported to be 2 × 10^–14 g. Since C. albicans contains approximately 37 fg of DNA, the qPCR assay theoretically has a detection limit of two yeast organisms.

Additional Comments

      The advantages of qPCR assays to veterinary practitioners include rapid analysis of a sample (i.e., 2 hours) and the potential ability to detect the presence of microbial DNA in cases of infectious endometritis where traditional culture was unsuccessful. In our experience, the time interval from sample submission to identification of a positive or negative result for pathogen DNA is about 6 hours, with sequencing and final reporting of the causative agent approximately 24–48 hours later. Early detection of an infection will allow practitioners to make informed clinical decisions regarding antimicrobial therapy days before traditional culture results would be available. An added advantage of qPCR is that the assay offers the possibility of monitoring bacterial or fungal DNA levels during antimicrobial therapy. A reduction or absence of microbial DNA, as determined by qPCR, during therapy would indicate progress toward resolution.

      A current limiting factor for qPCR analysis of equine samples is availability of the technology. Currently СКАЧАТЬ