High quality viral RNA for veterinary molecular diagnostic assays is essential for rapid pathogen identification and surveillance. The ability to perform high throughput viral RNA isolation from diverse samples is highly desirable to increase laboratory efficiency, reduce labor cost, and improve pathogen detection. Ambion’s MagMAX™ technology has been successfully used for viral RNA/DNA purification from a variety of animal tissues, as well as from blood, serum, plasma, milk, swabs (e.g., oral, cloacal), and feces. Viral RNA has also been successfully isolated from blood and oral swabs stored on Whatman® FTA® cards.
Sensitive Disease Pathogen Purification and Detection from Diverse Biological Samples
The MagMAX protocol was successfully employed for high throughput viral RNA isolation from avian tracheal and cloacal swab samples. The purified RNA was subsequently used for quantitative RT-PCR (qRT-PCR) to detect Exotic Newcastle Disease (END) during the 2002/2003 END outbreak in California, when more than 100,000 swab samples were processed. The performance of the MagMAX AI/ND* Viral RNA Isolation Kits was validated for END diagnosis at the USDA/National Veterinary Service Laboratory by comparing this protocol (Ambion beads, Figure 1) to a previously validated protocol (Competitor’s glass-filter, Figure 1). The results shown in Figure 1 demonstrate that similar, and often better, viral RNA recovery was obtained using the MagMAX protocol even when 10-fold less sample was used.
Figure 1. MagMAX™-96 AI/ND Viral RNA Isolation Kit Achieves 10X Greater Sensitivity than Competitor’s Kit. Clinical tracheal and cloacal swab samples were collected from poultry infected with California Exotic Newcastle Disease (END). RNA was isolated from each 50 µl sample using the MagMAX protocol or 500 µl sample using a protocol previously validated by the USDA/National Veterinary Service Laboratory. The results demonstrated that when starting with 10-fold less sample, viral RNA detection using the MagMAX isolated RNA was as sensitive or more sensitive than isolation of the larger sample with the traditional protocol. Data generated by Drs. S Hietala, B Crossley, and colleagues (California Animal Health & Food Safety Laboratory System, UC Davis).
MagMAX was compared to a filter column method (Kit Q) for nucleic acid isolation of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Mycoplasma hyopneumoniae (M. hyo.), Transmissible Gastroenteritis Virus (TGEV), and Swine Influenza Virus (SIV). The results in Figure 2 demonstrate the high efficacy and efficiency of MagMAX technology for swine pathogen nucleic acid isolation from diverse tissues, even with lower sample input. Similar or better pathogen detection sensitivity was obtained with MagMAX compared to the procedure from Kit Q.
Figure 2. MagMAX™ Viral RNA Isolation Kit Provides Highly Effective Pathogen Nucleic Acid Isolation From Diverse Sample Types. The efficiency of swine associated pathogen nucleic acid isolation using MagMAX technology was compared with filter columns from a competitor’s kit (Kit Q). For all experiments described below, purified nucleic acid elution volume was 30 µl for MagMAX samples and 60 µl for Kit Q samples. 25 µl qRT-PCR or qPCR were performed using 5 µl MagMAX eluent or 2.5 µl Kit Q eluent. Even with less sample input (see below), pathogen detection was as sensitive (equivalent Cts) or more sensitive (lower Cts) using nucleic acid isolated with the MagMAX Viral RNA Isolation Kit. (A) Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) RNA was isolated from 50 µl serum using the MagMAX Viral RNA Isolation Kit protocol or from 140 µl using the column procedure of Kit Q. For PRRSV RNA isolation from lung, approximately 2 g tissue (1 mm cubes) was placed into 35 ml Earle’s Balanced Salt solution and processed in a Stomacher® Lab System (Seward Ltd) at normal speed for 2 min. The resulting tissue lysate was centrifuged at 4500 x g for 30 min, and the supernatant (50 µl and 140 µl) was used for PRRSV RNA purification using the MagMAX Viral RNA Isolation Kit and Kit Q, respectively. (B) Mycoplasma hyopneumoniae (M. hyo.) DNA was isolated from lung using the protocol described in A. For Transmissible Gastroenteritis Virus (TGEV) RNA purification, 200 µl (1 mg) of fecal material was resuspended in 400 µl PBS and centrifuged at 5000 x g for 10 min at 4ºC. Supernatant (50 µl and 140 µl) was used for TGEV RNA purification using the MagMAX Viral RNA Isolation Kit and Kit Q, respectively. (C) Swine Influenza Virus (SIV) RNA was isolated from lung tissue using the protocol described in A. The following viral genes were detected: M = nucleoprotein gene target; H = hemagglutinin gene subtype target; N = neuraminidase gene subtype target. Data generated by Dr. K Harmon and colleagues (Iowa State University).
Isolate Viral RNA from Tissues Stored on Whatman FTA Cards
Whatman FTA technology provides an innovative method for sample collection and storage. Plant and animal tissues are applied directly to the FTA paper, where nucleic acids are instantly captured and stabilized and pathogens and nucleases are inactivated. Samples can be safely shipped or stored at room temperature. The remarkably simple sample collection process is ideal for collection of tissues containing various pathogens, especially since MagMAX Viral Kits have been optimized for viral RNA isolation from FTA-stored oral swabs and blood tissues.
West Nile Virus (WNV) RNA from infected birds was efficiently isolated from brain tissues and oral swabs stored on FTA cards using MagMAX Viral Kits. Brain tissues and their respective FTA stored oral swabs from 160 birds were tested; 12 WNV positive birds were identified correctly. The results in Figure 3A show highly sensitive (Ct values of 16–23) detection of WNV; many FTA samples yielded better sensitivity (lower Ct) than brain samples.
Figure 3. MagMAX™ Viral RNA Isolation Kits Effectively Isolate Viral RNA From Tissues Stored on Whatman® FTA® Cards. (A) West Nile Virus (WNV) was isolated from brain tissue homogenates and FTA-stored oral swabs using MagMAX-96 Viral RNA Isolation technology and the KingFisher® Magnetic Particle Processor (Thermo Electron Corp.). Brain tissues were homogenized as 10% solutions (0.1 g tissue/1 ml lysis/binding solution concentrate) and briefly centrifuged to clarify lysates. Homogenates (50 µl) were used for RNA purification with the MagMAX-96 Viral RNA Isolation Kit. FTA-stored oral swabs were punched into 2 mm spots and resuspended in MagMAX FTA Extraction Buffer, and 50 µl was used for RNA isolation with the MagMAX-96 Viral RNA Isolation Kit. Purified RNA was eluted using 50 µl Elution Buffer, and 5 µl of the purified RNA was used for 25 µl qRT-PCR. 100% concordance was observed between the two sample types; better detection sensitivity (lower Ct) was observed with many FTA stored samples. (B) 50 µl of Bovine Viral Diarrhea Virus persistently infected samples (19 PI samples) and negative blood samples (5 normal samples) were spotted on FTA cards. A 1 cm disk was excised from each blood spot, resuspended into 200 µl of MagMAX FTA Extraction Buffer, and vortexed moderately for 15 min at room temperature. The extract (50 µl) was used for RNA isolation with the MagMAX-96 Viral RNA Isolation Kit. Purified RNA was eluted in 30 µl Elution Buffer, and 2 µl of purified RNA was used for 10 µl qRT-PCR targeting BVDV RNA. Highly sensitive detection of BVDV RNA was achieved using RNA purified from FTA stored blood and whole blood. Data in Panel A generated by R Baker (Oregon State University Veterinary Diagnostic Laboratory)
Bovine Viral Diarrhea Virus (BVDV) RNA was also successfully isolated from BVDV positive blood stored on FTA cards. BVDV persistently infected (PI) samples (19 samples) and negative control samples (5 samples) were stored on FTA cards for 2 or 7 days at room temperature and used for viral RNA isolation. BVDV RNA recovery was assessed by qRT-PCR. The results in Figure 3B demonstrate effective and efficient BVDV RNA recovery. Blood (2 and 7 day old) stored on FTA cards yielded similar BVDV RNA recovery (similar Ct values of 24–28), while whole blood samples provided the most efficient BVDV RNA recovery (19–20 Ct values). In addition, approximately 5–8 Ct shifts were observed between corresponding whole blood and FTA samples. Taken together, these results indicate that FTA and MagMAX technologies are highly effective methods of pathogen sample collection and isolation.
This data was presented at the American Association of Veterinary Laboratory Diagnosticians (AAVLD) Annual Meeting. [Fang X, Bounpheng MA, Harmon K, Baker R, Mulrooney DM, Willis RC, Xu W, Hoang Q, Burrell AM (2005) Magnetic bead based high throughput viral RNA isolation enables superior animal pathogen molecular diagnostics. AAVLD, Hershey, PA, November 5–10.]
We thank CAHFS, Dr. Sharon Hietala, and especially Dr. Beate Crossley, for their assistance in testing and validating the MagMAX technology for high throughput END viral RNA isolation, and for providing and sharing their experimental results. Many thanks also to the USDA/NVSL Diagnostic Virology Laboratory for the validation of the MagMAX-96 Viral RNA Isolation Kit for END viral RNA isolation. We thank Karen Harmon’s laboratory at Iowa State University for testing MagMAX for swine related pathogen isolation and detection and Rocky Baker and Oregon State University Veterinary Diagnostic Laboratory for WNV isolation and detection data.
* MagMAX™ AI/ND Kits are the original kits that have been approved by the USDA/National Veterinary Service Laboratory for the isolation of viruses that cause avian influenza and Newcastle disease. (AI/ND = Avian Influenza/Newcastle Disease)