In January 2023, the laboratory of the Ministry of Health of Burkina Faso published an article "Comparative evaluation of automated KingFisher Flex Purification System 96 (ThermoFisher Scientific) and manual QIAamp Viral RNA Mini Kit ( Qiagen) extraction methods for SARS-CoV-2” on African Journal of Clinical and Experimental Microbiology.

This study explores the establishment of a new COVID-19 detection system, using Rainsure's COVID-19 extraction-free kit to compare the efficiency of two nucleic acid extraction platforms, Qiagen and Kingfisher, focusing on the rapid molecular detection of COVID-19 infection. And as an important clinical method for pathogen detection, to ensure rapid and accurate screening and diagnosis of patients with Covid-19 infection. The results of this study show that the automatic extraction method of KingFisher Flex Purification System 96 (ThermoFisher) is less sensitive and specific than the manual extraction method of QIAamp Viral RNA Mini Kit (Qiagen).

Title: Comparative evaluation of automated KingFisher Flex Purification System 96 (ThermoFisher Scientific) and manual QIAamp Viral RNA Mini Kit (Qiagen) extraction methods for SARS-CoV-2

Journal: "African Journal of Clinical and Experimental Microbiology"

Unit: Ministry of Health of Burkina Faso

Published: January 2023

Research Platform: Rainsure New Coronary Extraction Free Kit

Background

The nucleic acid extraction efficiency of SARS-CoV-2 specimens directly affects the quality of reverse transcription polymerase chain reaction (RT-PCR) test results, leading to errors in the diagnosis of new coronary pneumonia. The aim of this cross-sectional study was to evaluate the diagnostic performance of the automated extraction system KingFisher Flex Purification System 96 (ThermoFisher) compared with the manual extraction method of the QIAamp Viral RNA Mini Kit (Qiagen).

Method

From October to December 2020, specimens were collected from Burkina Faso's suspected imported cases or contacts of patients with Coronavirus, and 159 fresh nasopharyngeal swab specimens and 120 frozen nasopharyngeal swab specimens were tested. Rainsure’s FastPlexTM Triplex 1-Step COVID 19 Detection Kit (RT-PCR, RNA extraction free) was used to detect the products extracted by the two methods, and PCR amplification was completed in QuantStudio5 thermal cycler (Applied Biosystems). The analysis of the diagnostic performance of SARS-CoV-2 RT-PCR detection after RNA extraction by the two methods was completed using the online OpenEpi software (Fig. 1).

Figure 1: Flow chart of analysis steps for fresh (a) and frozen (b) samples

FastPlexTM Triplex 1-Step COVID 19 Detection Kit (RT-PCR, RNA extraction free) is used to amplify KingFisher Flex Purification System 96 (ThermoFisher) and QIAamp Viral RNA Mini Kit (Qiagen) on the same PCR plate ) two RNAs obtained by two methods, the gene targets of the amplification kit include: ORF1ab (FAM fluorescent dye) and N (HEX fluorescent dye) and internal reference (CY5 fluorescent dye) (Table 1).

Table 1: RT-PCR result table

Results

For fresh samples, the study found that the positive rate of RT-PCR after manual extraction was slightly higher than that of automatic extraction (12.6% vs 9.4%). For frozen samples, the positive rate of manual extraction was much higher than of automated extraction method, (38.33% vs 20.83%). The results showed that the automatic extraction method was less effective than manual extraction for fresh samples (sensitivity 35%, specificity 94.2%) and frozen samples (sensitivity 43.5%, specificity 93.2%). However, after McNemar's test with Yates correction, for fresh samples, there was no significant difference in the positive rate of RT-PCR between the two extraction methods (x2=0.76, p=0.38), while there was a significant difference in frozen samples (x2=12.9, p= 0.0003).

The study found that the RT-PCR positive rate of manual extraction of fresh samples (12.6%, 20/159) was slightly higher than that of automatic extraction (9.4%, 15/159). Seven samples (4.4%) were positive in both manual and automated extractions, while 131 samples (82.4%) were negative. Compared with manual extraction, the specificity of automatic extraction was 94.2%, the sensitivity was 35.0%, the PPV of fresh samples was 46.7%, and the NPV was 90.9%, but using McNemar's test with Yates correction, there was no difference between the two extraction methods for fresh samples. Significant difference (x2=0.76, p=0.38) (Table 2a).

Table 2a: Comparative evaluation of RT-PCR results after automatic and manual extraction of fresh samples

For frozen samples, there was a significant difference in the positive rate of RT-PCR, among which the manual extraction method was (38.33%, 46/120), and the automatic extraction method was (20.83%, 25/120) (x2=12.9, p=0.0003). Twenty samples (16.7%) were positive in both manual and automated extractions, while 69 samples were negative (57.5%). Compared with manual extraction, automatic extraction had specificity of 93.24%, sensitivity of 43.48%, PPV of 80.0%, and NPV of 72.63% (Table 2b).

Table 2b: Comparative evaluation of RT-PCR results after automatic and manual extraction of frozen samples

For fresh samples, the automatic extraction system KingFisher Flex Purification System 96 (ThermoFisher) compared with the QIAamp Viral RNA Mini Kit (Qiagen) manual extraction method, the amplification folds of ORF1ab and N genes were 2.36 and 0.81 times, respectively, which was statistically significant difference. However, there was no significant difference between the two in the average Ct value of the N gene (p=0.1319). In addition, there was no significant difference between the average Ct values of internal reference genes (p=0.4939) (Table 3).

Table 3: Average Ct values of SARS-COV-2 gene RT-PCR after extraction of fresh and frozen samples

Compared with the manual extraction method of QIAamp Viral RNA Mini Kit (Qiagen), the automatic extraction system KingFisher Flex Purification System 96 (ThermoFisher) has no significant difference in the average Ct value of ORF1ab (p=0.09885). Likewise, there was no significant difference between the mean Ct values of N genes between the two automatically extracted (p=0.0594). In addition, there was no significant difference between the average Ct values of the internal reference genes (p=0.1045) (Table 3).

Conclusion

The results of this study showed that the automated extraction system KingFisher Flex Purification System 96 (ThermoFisher) was less sensitive and specific than the QIAamp Viral RNA Mini Kit (Qiagen) manual extraction method. These data can be used as guidance information for laboratories to choose RNA extraction methods for RT-PCR detection of SARS-CoV-2.