Establishment of a Triple-primer Multiplex PCR Assay for Discriminative Detection of Duck and Goose Circoviruses
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摘要:
目的 建立一种可同时检测并鉴别鸭圆环病毒和鹅圆环病毒的PCR方法。 方法 根据已发布的鸭圆环病毒和鹅圆环病毒基因组分子特征设计3条引物，应用3条引物建立了一种在一个反应管中可检测并鉴别鸭圆环病毒和鹅圆环病毒的三引物PCR方法。 结果 该方法可特异性扩增鸭圆环病毒和鹅圆环病毒，并根据扩增片段大小鉴别病毒种类，检测最低限分别为110 、80 pg·μL−1。应用该方法对临床疑似病例样品进行检测，其结果与用2种病毒对应的单病毒PCR检测方法一致。 结论 建立的三引物PCR方法特异性强，敏感性高，丰富了水禽圆环病毒病的临床病例的诊断及流行病学监测的技术手段。 Abstract:Objective A PCR assay was established for discriminative detection of duck circovirus (DuCV) and goose circovirus (GoCV). Methods Three primers designed according to the genome sequences of DuCV and GoCV deposited in GenBank database were used for the development of a Triple-primer Multiplex PCR (tpm-PCR). Results The established assay could specifically identify DuCV and GoCV by the amplified fragments within the detection limits of 110 pg·μL−1 and 80 pg·μL−1, respectively. The methodology was verified with the test results by PCR for the individual virus in clinical cases. Conclusion The tpm-PCR assay was specific and sensitive as a discriminative detection method between DuCV and GoCV. It facilitated the clinical diagnosis and epidemiological monitoring on the circoviral cases in waterfowls. -
Key words:
- Duck circovirus /
- goose circovirus /
- triple-primer multiplex PCR /
- detection assay
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图 1 三引物PCR方法扩增及特异性电泳结果
M：分子量标准DL2 000；1：阴性对照；2：DuCV；3～4：GoCV; 5：鸭瘟病毒；6：番鸭细小病毒；7：鸭3型腺病毒；8：小鹅瘟病毒。
Figure 1. Amplification and specificity of tpm-PCR assay
M: Molecular marker DL2 000; 1: Negative control; 2: DuCV; 3–4: GoCV; 5: Duck plaque virus; 6: Muscovy parvovirus; 7: Duck adenovirus type 3; 8: Goose parvovirus.
图 2 PCR检测敏感性
M：分子量标准DL2 000；1～5分别为稀释1×10−1～1×10−5稀释。
Figure 2. Detection sensitivity of PCR method
M: Molecular marker DL2 000; 1–5: Dilutions of 1×10−1 to 1×10−5.
表 1 三引物PCR方法与其他方法检测比较
Table 1. Test results of different detection methods
样品
Samples样品数
Sample amount阳性数
Positive samples三引物PCR方法
Triple primers methodGoCV PCR方法
PCR assay for GoCVDuCV PCR方法
PCR assay for DuCV鹅 Goose 37 18（48.6%） 18（48.6%） - 鸭 Duck 56 22（38.6%） - 22（38.6%） 
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