A semiautomated extraction protocol of HCV-RNA using Favorgen RNA extraction kit has been developed. The kit provided protocol was modified by replacing manual spin steps with vacuum filtration. The assay performance was evaluated by real-time qPCR based on Taqman technology. Assay linearity was confirmed with the serial dilutions of RTA (Turkey) containing 1
The extraction process is a key component of nucleic acid detection, as it affects both the reliability and the reproducibility of target amplification. The manual extraction method (spin based) using Favorgen extraction RNA kit is time consuming and requires meticulous technical skills to achieve reproducible results [
Although the introduction of real-time PCR has led to considerable progress in automating the amplification and detection steps, still nucleic acid isolation remains very labor-intensive when performed manually. Thus, the objective of the present study was to evaluate performance of vacuum manifold system and spin based protocols for extraction of HCV-RNA using Favorgen kit high efficiency silica based spin column as reported in [
In the present study, 30 EDTA anticoagulated plasma samples submitted to the Punjab Institute of Nuclear Medicine for quantitative analysis of HCV were processed. HCV-RNA had been extracted by use of the Favorgen kit according to the manufacturer’s instructions [
Spin and vacuum filtration based protocol for HCV-RNA extraction using Favorgen kit.
To permit a higher throughput, the manual protocol described above was implemented on vacuum filtration assembly (Welch-Ilmvac 2522) under conditions as shown in Figure
HCV-RNA was quantified using real-time Taqman based AmpliSens HCV-FRT kits [
RT-qPCR was completed on Rotor-Gene Q 6000 (5 Plex-HRM) using the following cycling parameters: an initial cDNA synthesis by holding at 50 and 95°C for 15 min each, followed by 45 cycles of denaturation (95°C for 2 s), annealing (60°C for 5 s), and extension (72°C for 15 s). Mathematical analysis and graphical representations were performed using Rotor-Gene Q software.
Almost 30 clinical samples were subjected to RNA extraction in parallel by the spin and vacuum filtration based protocol using Favorgen kit. The experimental conditions of vacuum filtration were optimized by repeating experiments using positive control. RT-qPCR results by two extraction methods are given in Table
RT-qPCR results using spin and vacuum filtration based protocols for HCV-RNA extraction.
Sample ID |
CT |
CT | CT | IU mL−1 | IU mL−1 | Cv % |
---|---|---|---|---|---|---|
Vacuum filtration | Spin | Vacuum filtration | Spin | |||
1 | 25.99 | 15.6 ± 0.3 | 18.4 ± 0.4 | 481,419 | 67,458 | 0.9 |
2 | 26.48 | 16.0 ± 0.3 | 17.0 ± 0.3 | 360,304 | 177,932 | 0.6 |
3 | 26.57 | 22.6 ± 0.7 | 24.7 ± 0.5 | 3,602 | 847 | 0.01 |
4 | 26.8 | 12.8 ± 0.2 | 15.1 ± 0.3 | 3,299,175 | 704,535 | 6.4 |
5 | 27.5 | 18.6 ± 0.4 | 19.6 ± 0.4 | 60,629 | 28,823 | 0.1 |
6 | 26.34 | 13.3 ± 0.2 | 15.7 ± 0.3 | 2,461,014 | 430,986 | 4.7 |
7 | 27.14 | 16.1 ± 0.3 | 18.8 ± 0.4 | 348,791 | 51,043 | 0.7 |
8 | 26.86 | 17.6 ± 0.3 | 18.6 ± 0.4 | 112,812 | 56,348 | 0.2 |
9 | 27.84 | 14.6 ± 0.2 | 16.8 ± 0.3 | 946,830 | 206,548 | 1.8 |
10 | 26.54 | 17.2 ± 0.3 | 31.8 ± 1.2 | 160,482 | 54,132 | 0.3 |
11 | 26.29 | 19.2 ± 0.4 | 20.4 ± 0.6 | 38,131 | 16,544 | 0.1 |
12 | 24.9 | 21.4 ± 0.6 | 22.18 ± 0.7 | 9,683 | 5,736 | 0.0 |
13 | 24.7 | 15.07 ± 0.3 | 17.2 ± 0.3 | 767,124 | 179,381 | 0.2 |
14 | 26.28 | 18.9 ± 0.4 | 19.8 ± 0.4 | 52,693 | 29,976 | 0.02 |
15 | 26.77 | 22.2 ± 0.7 | 22.6 ± 0.7 | 5,810 | 4,340 | 0.0 |
16 | 26.2 | 20.4 ± 0.6 | 21.2 ± 0.6 | 19,312 | 11,584 | 0.01 |
17 | 25.4 | 19.8 ± 0.4 | 20.0 ± 0.4 | 29,849 | 25,136 | 0.01 |
18 | 26.8 | 21.6 ± 0.6 | 21.8 ± 0.7 | 8,328 | 7,507 | 0.00 |
19 | 26.1 | 15.3 ± 0.3 | 17.1 ± 0.3 | 642,090 | 187,782 | 0.2 |
20 | 26.0 | 21.4 ± 0.6 | 23.4 ± 0.7 | 9,533 | 2,555 | 0.0 |
21 | 26.1 | 18.8 ± 0.4 | 19.0 ± 0.4 | 55,731 | 50,456 | 0.02 |
22 | 26.1 | 17.2 ± 0.3 | 18.3 ± 0.4 | 171,609 | 83,106 | 0.06 |
23 | 25.7 | 19.3 ± 0.4 | 20.1 ± 0.4 | 46,845 | 24,354 | 0.02 |
24 | 28.4 | 21.5 ± 0.6 | 22.5 ± 0.7 | 9,201 | 4,760 | 0.0 |
25 | 26.1 | 22.1 ± 0.6 | 24.3 ± 0.7 | 5,980 | 1,307 | 0.0 |
26 | 26.8 | 20.3 ± 0.6 | 20.7 ± 0.6 | 20,985 | 16,260 | 0.01 |
27 | 31.5 | 26.0 ± 0.9 | 26.9 ± 0.8 | 280 | 150 | 0.01 |
28 | 18.5 | 18.1 ± 0.3 | 18.5 ± 0.4 | 95,495 | 70,695 | 0.04 |
29 | 31.2 | 16.2 ± 0.3 | 17.5 ± 0.3 | 352,282 | 145,151 | 0.1 |
30 | 27.5 | 19.44 ± 0.4 | 20.5 ± 0.6 | 37,850 | 18,256 | 0.01 |
A bivariate normal distribution fit between two assays gave diagonally distributed density ellipsoid with correlation of 95% between vacuum filtration and spin based protocols, indicating that both methods were efficient in the removal of inhibitory substance; see Figure
Bivariate fit between two assays.
Nevertheless, the modification of protocol from spin to vacuum filtration allowed extraction to be completed within 35 minutes. In addition, costs for consumables in semiautomated vacuum filtration based extraction reduced from $1 to 0.5 with reduced labor. At the same time, this protocol is more environmentally friendly due to reduced incineration of infected collection tubes. Thus, semiautomation is a more forward looking approach for nucleic acid purification together with real-time qPCR.
The performance of semiautomated vacuum filtration based extraction method was shown to permit a quick extraction process and accurate results for a quantitative assay of HCV-RNA. The method might be an alternative to an expensive full automation station for developing countries which is easy to perform and efficient. In short, costly instruments are not required to prevent contamination and to enhance the safety of worker.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Imran Amin and Tania Jabbar have equal contributions and the names are written alphabetically.