Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR.
Development of a reference standard for the detection and quantification of Mycobacterium avium subsp. paratuberculosis by quantitative PCR.
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开发用于检测和定量分枝杆菌亚种的参考标准。通过定量PCR进行旁结核。
DOI：
10.1038/s41598-021-90789-0
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发表时间：
2021-06-02
影响因子：
4.6
通讯作者：
Kralik P
中科院分区：
文献类型：
--
作者：
Beinhauerova M;Beinhauerova M;McCallum S;Sellal E;Ricchi M;O'Brien R;Blanchard B;Slana I;Babak V;Kralik P
定量PCR（qPCR）已成为检测禽分枝杆菌禽亚种（Mycobacterium avium subsp.）副结核病（MAP）。然而，通过qPCR测定的MAP的量可能受到所用qPCR定量标准品的类型（PCR产物、质粒、基因组DNA）和测定标准DNA量的方式（吸光度、荧光）的影响。在实践中，这可能反映在无法正确比较来自不同实验室的相同qPCR测定的定量数据。因此，本研究的目的是制备国际MAP参比标准品的原型，可用于校准各实验室常规使用的qPCR定量标准品，以促进临床数据的可比性。考虑到稳定性、储存和运输问题，选择人工污染MAP参比菌株的冻干粪便混悬液作为标准品的最合适形式。通过F57 qPCR，以2周间隔监测5种类型冻干基质对标准品稳定性的影响，持续4个月。含10%脱脂乳的冻干基质提供了最佳的回收率和稳定性，因此被选择用于随后涉及6个诊断和研究实验室的标准品比较试验，其中DNA分离和qPCR测定程序与平行使用相同的供应基因组DNA溶液进行。此外，检测了储存条件对标准品稳定性的影响，持续至少6个月。在室温、黑暗和光照下、+4 °C、-20 °C和-80 °C下的储存显示稳定性没有显著变化，并且使用噬菌体扩增测定也没有发现MAP活力的实质性变化。制备的MAP定量标准品提供了均匀且可重现的结果，证明其适合用作国际参考qPCR标准品。
Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, − 20 °C and − 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.
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通过液滴数字 PCR 检测循环结核分枝杆菌特异性 DNA，用于受攻击猴子的疫苗评估和结核病诊断
影响因子：
13.2
作者：
Song N;Tan Y;Zhang L;Luo W;Guan Q;Yan MZ;Zuo R;Liu W;Luo FL;Zhang XL
通讯作者：
Zhang XL
影响因子：
4.4
作者：
Elguezabal, Natalia;Bastida, Felix;Juste, Ramon A.
通讯作者：
Juste, Ramon A.
影响因子：
1.8
作者：
Kralik, Petr;Beran, Vladimir;Pavlik, Ivo
通讯作者：
Pavlik, Ivo
影响因子：
2.7
作者：
Saldanha, J;Gerlich, W;Heath, A
通讯作者：
Heath, A
影响因子：
3.3
作者：
Kawaji, Satoko;Taylor, Deborah L.;Whittington, Richard J.
通讯作者：
Whittington, Richard J.