{"id":3121,"date":"2024-07-01T11:58:25","date_gmt":"2024-07-01T03:58:25","guid":{"rendered":"https:\/\/opentrons.com.cn\/?post_type=knowledge2&p=3121"},"modified":"2024-09-02T18:28:23","modified_gmt":"2024-09-02T10:28:23","slug":"lzyyrnatqfapg","status":"publish","type":"post","link":"https:\/\/en.opentrons.com.cn\/news\/lzyyrnatqfapg\/","title":{"rendered":"Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection"},"content":{"rendered":"\n
Two automated in-house protocols for high-throughput RNA extraction from nasopharyngeal swabs for the detection of SARS-CoV-2 have been evaluated.<\/p>\n\n\n\n
141 SARS-CoV-2 positive samples were collected over 10 days. The in-house protocol is based on magnetic bead extraction and is designed for use with the Opentrons OT-2 (OT-2Internal<\/sub>) liquid handling robot or the MagMAX Express-96 system (MMInternal ). Both protocols were tested in parallel with a commercial kit using the MagMAX TM system (MM Kit). Nucleic acid extraction efficiency was calculated based on the SARS-CoV-2 DNA positive control.<\/sub><\/p>\n\n\n\n No significant differences were found between in-house protocols and commercial kits in detecting positive samples. The MM reagent kit<\/sub> was the most efficient, although the internal MM kit<\/sub> had lower average Ct values \u200b\u200bthan the other two kits. Compared to commercial kits, the in-house protocol saves \u20ac350 to \u20ac400 per 96 samples extracted.<\/p>\n\n\n\n The described protocol utilizes readily available reagents and open source liquid handling systems and is suitable for SARS-CoV-2 detection in high-throughput facilities.<\/p>\n\n\n\n Citation:<\/strong> L\u00e1zaro-Perona F, Rodriguez-Antol\u00edn C, Alguacil-Guill\u00e9n M, Guti\u00e9rrez-Arroyo A, Mingorance J, Garc\u00eda-Rodriguez J, et al. (2021) Evaluation of two automated low-cost RNA extraction protocols for SARS-CoV-2 detection. PLoS ONE 16(2):e0246302. https:\/\/doi.org\/10.1371\/journal.pone.0246302<\/p>\n\n\n\n Editor:<\/strong> AM Abd El-Aty, Cairo University, Egypt<\/p>\n\n\n\n Received:<\/strong> November 5, 2020; Accepted:<\/strong> January 15, 2021; Published:<\/strong> February 16, 2021 day<\/p>\n\n\n\n Copyright:<\/strong> \u00a9 2021 L\u00e1zaro-Perona et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.<\/p>\n\n\n\n Data availability:<\/strong> All relevant data are within the paper and its supporting information files.<\/p>\n\n\n\n Funding:<\/strong> The authors received no specific financial support for this work.<\/p>\n\n\n\n Competing Interests:<\/strong> The authors declare that there are no competing interests.<\/p>\n\n\n\n The SARS-CoV-2 outbreak requires the large-scale use of qPCR testing to detect positive cases and trace contacts to stop community transmission. Before qPCR detection, RNA extraction from clinical samples is usually required [ 1-4 ]. Considering the number of samples tested daily, manual RNA extraction methods are not feasible for most institutions, therefore, automated systems are widely used for this task [5-7]. The disadvantage of an automated system is that it significantly increases the final cost, which may hinder large-scale testing in some areas. Additionally, inventory shortages of extraction reagents caused significant delays in diagnosis due to increased demand.<\/p>\n\n\n\n This article describes two low-cost automated RNA extraction methods. The first method uses the OT-2 system (Opentrons, New York, NY, USA), an open source liquid handling robot capable of automating self-designed protocols; the second method uses MagMAX, a fast and easy-to-use nucleic acid extractor. TM<\/sup> Express-96 system (Thermo Fisher Scientific, Waltham, MA, USA). The latter can extract up to 96 samples in 30 minutes, but requires prior manual distribution of reagents, magnetic beads, and samples in the 96-well plate, which adds 30 minutes. As an alternative, the OT-2internal<\/sub> protocol can process up to 48 samples in 104 minutes, fully automated.<\/p>\n\n\n\n Over ten days, 141 consecutive SARS-CoV-2-positive nasopharyngeal swabs with viral transport medium (Deltalab, Barcelona, \u200b\u200bSpain) were collected and stored at 4\u00b0C. Before processing, 500 \u03bcL of virus culture medium and 500 \u03bcL of 4M guanidine isothiocyanate (GTC) (Qiagen, Hilden, Germany) were mixed with 5 \u03bcg\/mL carrier RNA to inactivate the samples, and then the samples were incubated at 80\u00b0C. Heat for 2 minutes and vortex briefly to mix.<\/p>\n\n\n\n Two systems were used for automatic extraction of nucleic acids: MagMAX TM<\/sup> Express-96 deep well magnetic particle processor (King Fisher Instrument, Thermo Fisher Scientific, Waltham, MA, USA) and open system OT-2 ( Opentrons, New York, NY, USA) with GEN1 magnetic module (Opentrons, New York, NY, USA) and internal protocol. Both systems used MagMAX\u2122 Express 96 plates and deep well plates (Thermo Fisher Scientific, Waltham, MA, USA).<\/p>\n\n\n\n Three methods were used for nucleic acid extraction: 1) MagMAX and the commercial MagMAX CORE Nucleic Acid Purification Kit (MM Kit) (Thermo Fisher Scientific, Warburg, MA, USA) were used according to the manufacturer's instructions. Elsheim); 2) The OT-2 system uses universal reagents (inside OT-2), such as ethanol (Emsure\u00ae, Merck KGaA, Darmstadt, Germany), 2-propanol (Emsure\u00ae, Merck KGaA, Darmstadt, Germany), elution buffer (Omega BIO-TEK, Norcross, GA, USA), nuclease-free water (Ambion TM<\/sup>, Thermo Fisher Scientific, MA, USA) Waltham) and magnetic beads (Mag-Bind\u00ae TotalPure NGS, Omega Bio-Tek, Norcross, GA, USA); 3) MagMAX uses the same protocol as the commercial kit, but uses reagents from the OT-2 method (MM in-house >). The in-house protocol was a modified version of the procedure described by Hui He et al. [8]. Briefly, inactivated respiratory samples were mixed with isopropyl alcohol in a 1:1 ratio to a final volume of 500 \u03bcl (Internal OT-2) and 1000 \u03bcL (Internal<\/sub>MM), Add 40 \u03bcL of magnetic beads and incubate at room temperature for 5 minutes. Next, use a magnet to pull the beads to the side of the tube and discard the supernatant. The beads were then washed twice with 500 \u03bcL of freshly prepared 70% ethanol. After the second wash, discard the 70% ethanol and air-dry the beads at room temperature. Finally, the beads were resuspended in 100 \u03bcL elution buffer and separated again with a magnet to recover the eluted viral RNA (Table 1).<\/p>\n\n\n\n Table 1. Steps, reagents, and volumes (\u03bcL) used in the three protocols evaluated.<\/p>\n\n\n\n MMKit<\/sub> protocol:<\/p>\n\n\n\n MMinternal<\/sub> protocol:<\/p>\n\n\n\n OT-2internal<\/sub> protocol:<\/p>\n\n\n\n The OT-2 internal protocol is written in Python according to Opentrons instructions. The script is available in a GitHub repository<\/p>\n\n\n\n To validate the performance of the in-house nucleic acid extraction protocol, simulations of standard, low, and very low viral loads were produced using the DNA Positive Control TaqMan 2019-nCoV Control Kit v1 (Thermo Fisher Scientific, Waltham, MA, USA) sample. The concentration of the control kit is 1 x 10 4<\/sup> copies\/\u03bcL. Standard viral load samples were prepared by mixing 10 \u03bcL of positive control with 490 \u03bcL of viral transport medium and 500 \u03bcL of GTC. Low and very low viral loads were prepared in the same manner but using tenfold serial dilutions of the positive control. All simulated samples were prepared in triplicate and processed in parallel with Internal<\/sub>OT-2, MMKit and<\/sub>Internal<\/sub>MM, and then used TaqMan 2019- nCoV detection kit v1 for qPCR testing. The final concentrations of positive controls in standard, low and very low viral load mock samples were 1 x 10 6<\/sup> copies\/mL, 1 x 10 5<\/sup> copies\/mL and 1 x 10 4<\/sup>copies\/mL. All runs included negative controls.<\/p>\n\n\n\n Calculate nucleic acid extraction efficiency by comparing the Ct value of the positive control in the simulated sample (Ct ss<\/sub> ) with the Ct value of the positive control prepared directly from stock, correcting the amount to match the dilution factor and each protocol The initial sample size used in (Ct pc<\/sub> ) (R = 2 -\u0394Ct<\/sup> = 2 -(Ctss-Ctpc)<\/sup> ).<\/p>\n\n\n\n Using TaqMan 2019-nCoV detection kit v1 targeting orf1ab<\/em>, spike (S), nucleocapsid (N) and human RNaseP genes and TaqMan 2019-nCoV control kit v1 as a positive control, Nucleic acid amplification of SARS-CoV-2 viral RNA was performed according to the qPCR conditions recommended by the manufacturer. All qPCR assays were performed in a CFX96 Touch real-time PCR detection system (Bio-Rad, Hercules, CA, USA). To reduce inter-assay variability, extracted nucleic acid samples are automatically dispensed into qPCR test strips using another OT-2 module.<\/p>\n\n\n\n The three alternatives were compared in pairs. McNemar's test was used to compare their performance in assigning samples as positive or negative. Ct values \u200b\u200bwere not normally distributed, so the Wilcoxon signed-rank test was used to compare Ct values \u200b\u200bfor each target. For each target, only samples amplified by these three methods were considered. Use the bootstrap method to calculate mid-position confidence intervals.<\/p>\n\n\n\n All statistical tests were performed using the IBM SPSS Statistics 24.0.0.0 software package (SPSS Inc., Chicago, IL, USA).<\/p>\n\n\n\n The nucleic acid extraction efficiency of the two in-house protocols was compared with the MMkit protocol by extracting mock samples prepared with SARS-CoV-2 positive controls simulating different viral loads. MM<\/sub>Kit<\/sub> and OT-2 In-house<\/sub> protocols successfully amplified orf1ab<\/em> in all simulated samples with standard viral loads , S and N targets. For these samples, the average extraction efficiency across all replicates and genes was 33.9% (SD: 13.8) for the MM kit and 19.6% (SD: 2.2) for the OT-2 in-house protocol. , MMInternal Plan<\/sub>16.0% (SD: 5.03).<\/p>\n\n\n\n In the low viral load simulated samples, the MM kit<\/sub> was unable to amplify the S target in all samples, nor the N target in one of the replicate samples, while the OT-2 internal <\/sub> and MMinternal<\/sub> detected all genes. Finally, the MMkit<\/sub> and OT-2in-house<\/sub> protocols were unable to amplify very low viral load samples, except for one duplicate sample in which the N gene could be amplified by OT-2Internal<\/sub> protocol amplification, but MMInternal<\/sub> could detect the positive control in all samples, one containing three targets and one containing orf1aband<\/em> N target, the last sample contained theorf1ab<\/em>target (S1 Table).<\/p>\n\n\n\n The 141 positive clinical samples collected were simultaneously extracted using MM kit<\/sub>, OT-2internal<\/sub> method and MMinternal<\/sub> method, and analyzed by qPCR Detection of eluted SARS-CoV-2 RNA. Positive\/negative results and amplification cycle threshold (Ct) were recorded for each target. Samples were considered positive when at least one of the three target regions was amplified and Ct<40, according to the manufacturer's instructions. Negative controls included in all runs produced negative PCR results in all cases.<\/p>\n\n\n\n Of the 141 samples, 123 tested positive for SARS-CoV-2 by at least one extraction method, while 18 samples tested negative by all three methods. This may be due to degradation of the samples during collection since they had been stored at 4\u00b0C for approximately 48 h [9]. By method, 114 samples tested positive using the MM kit, 111 samples tested positive using the in-house<\/sub>OT-2, and 118 samples tested positive using thein-house<\/sub>MM . Pairwise comparison found no significant difference in their performance in detecting SARS-CoV-2 (MMKit<\/sub>Vs OT-2Internal<\/sub>, P =<\/em> 0.5465 ; MMKit<\/sub>Vs MMInternal<\/sub>, P =<\/em> 0.3865; OT-2Internal<\/sub>Vs MMinternal<\/sub>, P =<\/em> 0.0961). Eighteen samples tested negative by some of the three methods. These had higher Ct values, with median values \u200b\u200bof 38.68 and 38.19 for the orf1ab<\/em> and N targets, respectively (in these samples, the S target was not amplified by any method). Pairwise linear regression and correlation analyzes comparing all methods and targets showed R2<\/sup> values \u200b\u200bbetween 0.85 and 0.95, and Bland-Altman analysis showed consistent agreement across the entire Ct range (S1 Fig ).<\/p>\n\n\n\n In 43% of the samples extracted using the MM kit<\/sub>, three targets (orf1ab, N and S) were detected, and 43% of the samples extracted using the MM kit<\/em>Internal<\/sub> It was detectable in 49% of samples extracted with OT-2 and in 49% of samples extracted using in-house MM (Figure 1). orf1ab<\/em>and the N target were detected in 34%, 39%, and 30% of samples, and only the N target was amplified in 19%, 10%, and 17% of samples. Only a very small number of samples were considered positive due to amplification of the orf1ab<\/em> target alone (6), the orf1ab<\/em> + S (1) or the N + S (5) target, and no The sample had the S gene as the only positive marker. In fact, the S target in this assay clearly lacks sensitivity and is irrelevant to diagnostic decisions. Two or three targets were detected in 87% (97 samples) of samples using the OT-2internal<\/sub> protocol and two or three targets were detected using the MMinternal protocol. 82% (95 samples), and 79% (90 samples) of samples with two or three targets detected using the MM kit.<\/sub><\/p>\n\n\n\nresult<\/h3>\n\n\n\n
in conclusion<\/h3>\n\n\n\n
introduce<\/h2>\n\n\n\n
method<\/h2>\n\n\n\n
Sample collection<\/h3>\n\n\n\n
Equipment and reagents<\/h3>\n\n\n\n
<\/a><\/figure>\n\n\n\n
Protocol design and verification<\/h3>\n\n\n\n
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The sample input volume is the maximum allowed by the automatic pipetting system. The volumes of other reagents are also different, so the input volumes for the three methods are different.<\/h3>\n\n\n\n
quantitative PCR<\/h3>\n\n\n\n
Statistical analysis<\/h3>\n\n\n\n
result<\/h2>\n\n\n\n
The efficiency of the extraction plan<\/h3>\n\n\n\n
Nucleic acid extraction from clinical respiratory samples<\/h3>\n\n\n\n