“Swine flu” is caused by the swine influenza virus (A/H1N1). This virus was first discovered in 1930 in pig populations. The recently discovered subtype A of the H1N1 virus includes genes derived by reassortment from human, swine, and avian viruses, which can now be transmitted from human to human. As with all influenza infections, reliable and sensitive detection of the virus is an important prerequisite for identifying suspicious patients and supporting infection control. Rapid tests for influenza virus detection that use antibodies to detect virus proteins are available and commonly used. However, these tests are often less sensitive than real-time RT-PCR-based methods and are prone to false negative results. Therefore, RT-PCR based methods are considered the gold standard. Typically, swabs or nasopharyngeal swab samples are taken from patients and used for viral RNA extraction. Demand is high for RNA extraction methods that offer sensitivity and reproducibility. Moreover, since it is usually necessary to screen a large number of samples, extraction is typically automated using a high-throughput method.

In this application note, the magnetic bead-based NucleoMag Virus kit was used in combination with the Thermo KingFisher Flex magnetic particle separator to purify viral RNA from swabs that were washed and incubated in NaCl solution. The method starts with a lysis incubation of the swab wash solution and subsequently, after addition of binding buffer, the released nucleic acids are bound to magnetic particles. Following several washing steps, the purified viral RNA is eluted and can be used for real-time PCR (RT-PCR) downstream applications. The KingFisher Flex separation device allows the purification of up to 96 samples in approximately 60 min.

RT-PCR analysis of influenza virus A/H1N1 RNA isolated from INSTAND interlaboratory test samples

Two different RT-PCR systems were used to evaluate the quality of influenza virus A/H1N1 RNA isolated from INSTAND interlaboratory test samples using the NucleoMag Virus kit in combination with the Thermo KingFisher Flex magnetic particle separator. Real-time RT-PCR amplification was performed according to the recommendations of the Robert Koch Institute (RKI) or using a commercial detection kit (the Influenza Screen & Type RT-PCR Kit 1.0 kit from Astra Diagnostics).

RT-PCR analysis according to Robert Koch-Institute (RKI) recommendations

RT-PCR analysis according to Robert Koch Institute (RKI) recommendations uses two independent master mixes. One RT-PCR reaction detects influenza A viruses, while the second PCR reaction is specific for the H1N1 subtype. Consequently, a sample which is positive for H1N1 should be positive in both PCR reactions. Influenza A subtypes different from H1N1 should yield only positive signals in the influenza A reaction. The results are summarized in Table 1.

RT-PCR analysis using the Influenza Screen & Type RT-PCR Kit 1.0 kit from Astra Diagnostics

This kit uses a master mix for the detection of both influenza A and the H1N1 subtype (the primers bind to similar target sequences). Only one of the primers (either influenza A or H1N1) can bind to the target. Therefore, only one signal is generated: one PCR reaction detects influenza A viruses, while the second PCR reaction is specific for subtype H1N1. Consequently, a sample which is positive for H1N1 should be negative for influenza A. Influenza A subtypes different from H1N1 should yield only positive signals in the influenza A reaction. The results are summarized in Table 2.

As seen in Tables 1 and 2 with both PCR systems, all samples from the interlaboratory test were identified correctly. No false positives or false negatives were observed.

*Real-time PCR was performed according to the recommendations of the Robert Koch-Institute (RKI). The virus purification protocol used is summarized in Table 3 in Methods.

*Real-time PCR was performed using the Influenza Screen & Type RT-PCR Kit 1.0 kit from Astra Diagnostics. The virus purification protocol used is summarized in Table 3 in Methods.

RT-PCR analysis of influenza virus A/H1N1 RNA isolated from clinical samples

Viral RNA was isolated from swab samples obtained from clinical patients using the NucleoMag Virus kit and subjected to RT-PCR specific for either influenza A or H1N1. RT-PCR setup was performed according to the recommendations of the Robert Koch-Institute (RKI) on a Roche LightCycler 480 instrument. Amplification plots are shown in Figure 1 (influenza A) and Figure 2 (H1N1).

Analysis of Amplification Data

Calculated CP values are summarized in Figure 3. All samples which tested positive for the presence of the H1N1 virus (Figure 3, top) also tested positive for influenza A (Figure 3, bottom), clearly identifying the samples positive for the swine virus A/H1N1. All samples which tested negative for H1N1 also tested negative for influenza A. In both RT-PCR systems, the negative controls consistently tested negative, while the positive controls consistently tested positive.

Two different RT-PCR systems were used to correctly identify all the samples from an interlaboratory test, which were purified using the NucleoMag Virus kit. No false positives or false negatives were observed. Consistent results were also obtained for clinical swab samples purified using the NucleoMag Virus kit. These results indicate that this kit yields high-quality viral RNA suitable for downstream RT-PCR applications. 

Viral RNA isolation

The kit evaluation was performed by Labcon OWL, Bad Salzuflen, Germany, with clinical samples and INSTAND interlaboratory test samples.

The following samples were used in the study:

• INSTAND round-robin samples for influenza A/H1N1. Samples are suitable for the detection of human influenza A and B viruses as well as new influenza A subtype H1N1 (swine flu).
• Samples are provided as lyophilized lysates of tissue culture media or allantoic liquid from infected and incubated hens' eggs. 
• Samples were extracted with the NucleoMag Virus kit automated on a KingFisher Flex magnetic separator. After elution, aliquots of the eluates were used in conjunction with two different PCR systems for the detection of influenza A and A/H1N1.

In addition to the samples from the interlaboratory test, swab samples from potentially positive patients were subjected for viral RNA isolation.

The purification protocol used is summarized in Table 3.

RT-PCR

RT-PCR was performed according to Robert Koch Institute (RKI) recommendations (Lightcycler 480) or using the Influenza Screen & Type RT-PCR Kit 1.0 kit from Astra Diagnostics (Hamburg, Germany).

Robert Koch Institut, www.rki.de. TaqMan real-time PCR for detection of porcine influenza A/H1N1-virus