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客戶采用我司硅羥基磁珠研究低載量新冠病毒檢測技術在Analytica Chimica Acta發文

2022-3-9 22:51:40點擊:

客戶采用我司硅羥基磁珠研究低載量新冠病毒檢測技術在Analytica Chimica Acta發文

Importance of sample input volume for accurate SARS-CoV-2 qPCR testing
Yugan He,Tie Xie,Qihang Tu,Yigang Tong
https://doi.org/10.1016/j.aca.2022.339585


Abstract
Nucleic acid testing is the most widely used detection method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic. Currently, a number of COVID-19 real-time quantitative reverse transcription PCR (qPCR) kits with high sensitivity and specificity are available for SARS-CoV-2 testing. However, these qPCR assays are not always reliable in detecting low viral load samples (Ct-value ≥ 35), resulting in inconclusive or false-negative results. Here, we used a Poisson distribution to illustrate the inconsistent performance of qPCR tests in detecting low viral load samples. From this, we concluded that the false-negative outcomes resulted from the random occurrences of sampling zero target molecules in a single test, and the probability to sample zero target molecules in one test decreased significantly with increasing purified RNA or initial sample input volume. At a given RNA concentration of 0.5 copy/μL, the probability of sampling zero RNA molecules decreased from 36.79% to close to 0.67% after increasing the RNA input volume from 2 to 10 μL. A SARS-CoV-2 qPCR assay with an LOD of 300 copies/mL was used to validate the improved consistency of the qPCR tests. We found that the false-negative qPCR results of clinical COVID-19 samples with a Ct ≥ 35 decreased by 50% after increasing the input of purified RNA from 2 to 10 μL. The consistency, accuracy, and robustness of nucleic acid testing for SARS-CoV-2 samples with low viral loads can be improved by increasing the sample input volume.

核酸檢測是嚴重急性呼吸系統綜合癥冠狀病毒 2 (SARS-CoV-2) 最廣泛使用的檢測方法,它是 2019 年冠狀病毒病 (COVID-19) 大流行的病原體。目前,許多具有高靈敏度和特異性的 COVID-19 實時定量逆轉錄 PCR (qPCR) 試劑盒可用于 SARS-CoV-2 檢測。然而,這些 qPCR 檢測在檢測低病毒載量樣本(Ct 值 ≥ 35)時并不總是可靠的,從而導致不確定或假陰性結果。在這里,我們使用泊松分布來說明 qPCR 測試在檢測低病毒載量樣本中的不一致性能。由此,我們得出結論,假陰性結果是由于在單次測試中隨機發生零靶分子采樣,并且在一次測試中采樣零靶分子的概率隨著純化 RNA 或初始樣本輸入量的增加而顯著降低。在 0.5 拷貝/μL 的給定 RNA 濃度下,在將 RNA 輸入量從 2 增加到 10 μL 后,采樣零 RNA 分子的概率從 36.79% 下降到接近 0.67%。 LOD 為 300 拷貝/mL 的 SARS-CoV-2 qPCR 測定用于驗證 qPCR 測試的一致性改進。我們發現,在將純化 RNA 的輸入量從 2 μL 增加到 10 μL 后,Ct ≥ 35 的臨床 COVID-19 樣本的假陰性 qPCR 結果降低了 50%。通過增加樣本輸入量,可以提高病毒載量較低的 SARS-CoV-2 樣本核酸檢測的一致性、準確性和穩健性。


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