Verdin E, Kobisch M, Bové J M, Garnier M, Saillard C
UMR GDPP, Laboratoire de Biologie Cellulaire et Moléculaire, IBVM, Centre INRA de Bordeaux, 33883 Villenave dOrnon, France.
Mol Cell Probes. 2000 Dec;14(6):365-72. doi: 10.1006/mcpr.2000.0326.
We have previously reported a nested PCR assay for the detection of Mycoplasma hyopneumoniae directly in tracheobronchiolar washings from living pigs in field conditions. Here, we describe the construction and use of an internal control to monitor the presence of PCR inhibitors. A PCR modified target DNA was constructed by insertion of a small DNA fragment into the M. hyopneumoniae specific DNA target. We have demonstrated that the internal control failed to be amplified in only three tracheobronchiolar washings samples out of the 362 tested. This control molecule was inserted in a Spiroplasma citri derived plasmid vector and introduced into S. citri cells by electroporation. After a few passages we ensured that the recombinant plasmid became inserted into the genome of S. citri. PCR amplification of the DNA of this transformed S. citri strain using nested PCR primers led to amplification of a 900-bp fragment which can be discriminated from the M. hyopneumoniae PCR product 700 bp. The S. citri transformants with the integrated internal control were added to the tracheobronchiolar washings prior to PCR and used as an internal control to check the efficiency of sample processing, and to demonstrate the presence of inhibitors. Furthermore, we have been able to estimate the number of mycoplasma cells in the tracheobronchiolar washings. Quantitation was performed by comparing the PCR signal intensity of the specific M. hyopneumoniae template with known concentrations of the S. citri competitor. The titer in tracheobronchiolar washings ranged approximatively from 10(4)to 10(8)M. hyopneumoniae cells per ml of clinical specimen. Quantitative PCR can be a useful tool for monitoring the progression of M. hyopneumoniae in the disease process.
我们之前报道过一种巢式PCR检测方法，可直接在野外条件下对活猪的气管支气管冲洗液中的猪肺炎支原体进行检测。在此，我们描述了一种内部对照的构建及应用，用于监测PCR抑制剂的存在情况。通过将一个小DNA片段插入猪肺炎支原体特异性DNA靶标中，构建了一个经修饰的PCR靶标DNA。我们已证明，在362份检测的气管支气管冲洗液样本中，只有3份样本中的内部对照未能扩增。该对照分子被插入到一种源自柑桔螺原体的质粒载体中，并通过电穿孔法导入柑桔螺原体细胞。经过几代传代后，我们确保重组质粒已插入柑桔螺原体的基因组中。使用巢式PCR引物对这种转化后的柑桔螺原体菌株的DNA进行PCR扩增，可得到一个900 bp的片段，该片段可与猪肺炎支原体PCR产物的700 bp片段区分开来。在PCR之前，将带有整合内部对照的柑桔螺原体转化体添加到气管支气管冲洗液中，并用作内部对照，以检查样本处理的效率，并证明抑制剂的存在。此外，我们还能够估计气管支气管冲洗液中支原体细胞的数量。通过将猪肺炎支原体特异性模板的PCR信号强度与已知浓度的柑桔螺原体竞争物进行比较来进行定量。气管支气管冲洗液中的滴度范围约为每毫升临床标本含10⁴至10⁸个猪肺炎支原体细胞。定量PCR可成为监测猪肺炎支原体在疾病进程中进展情况的有用工具。
