Chapter 2: LITERATURE REVIEW
2.9 Proposed Research Methodology
2.9.6 Using RT-PCR to detect AHS
A number of researchers have developed PCR assays to detect the AHS virus since the early 1990‟s and a brief overview of their experimental designs and differences are described below.
2.9.6.1 Isolation of AHSV genomic material
The double stranded RNA genome of the AHS virus that is the target of a PCR would ordinarily need to be isolated from the virus structure and surrounding biological components. In some cases it may be possible to perform a PCR on crude samples which would drastically reduce the cost of the test (Watson, Personal Communication).
Stone-Marschat et al. (1994) purified viral dsRNA from infected Vero cell lysates by phenol extraction and lithium chloride precipitation based on the method of Clarke and McCrae (1981). Zientara et al. (1995b) isolated total RNA from cell cultures and spleen tissue samples using the guanidinium-thiocyanate-phenol-chloroform method of Chomczynski & Sacchi (1987). Commercial kits based on this method are now available such as the TRIzol™ group of reagents (Rodriguez-Sanchez et al., 2008a).
The quantity of AHSV genomic dsRNA can be determined spectrophotometrically at 260 nm (Wade-Evans et al., 1990).
In terms of field samples, it has been reported that the AHS viral genome was successfully extracted and purified from clotted blood. This has the potential to reduce costs further since special blood collection vials containing anti-coagulants may no longer be needed (Fasina, 2008; Fasina et al., 2008). In addition, direct PCR from whole blood may be possible by modifying the initial denaturation steps (Mercier et al., 1990; McCusker et al., 1992).
However, the possibility exists to eliminate viral RNA extraction procedures by using specially prepared filter paper (FTA® cards). In 1997, this was achieved with the Human Immunodeficiency Virus (HIV) Type 1. In addition, RNA levels on the filter paper had not decreased after two weeks at 20°C and three days at 37°C. This has important consequences for field studies and the development of field applicable diagnostic assays (Cassol et al., 1997). FTA cards have also been examined and for their ability to inactivate pathogens and for their storage ability and stability of nucleic acids, all of which revealed promising results for reducing field assay costs (Roy &
Nassuth, 2005; Purvis et al., 2006). In 2007, real-time RT-PCR was performed on RNA porcine reproductive and respiratory virus using FTA® cards (Inoue et al., 2007). By using a higher pH PCR buffer, Bu et al. (2008) were able to amplify genomic DNA directly from blood that had dried on the filter paper. This has an enormous potential to reduce costs involved in such diagnostic assays, as expensive reagents to extract genomic material will no longer be needed.
The extraction of viral RNA from the OBP manufactured freeze-dried vaccines may also be necessary. This has been achieved previously, albeit with an avian dsRNA reovirus (Bruhn et al., 2005)
2.9.6.2 Selection of primers
Short, complementary DNA sequences, or oligonucleotides, designed to anneal to target DNA are termed primers. Primers are selected to amplify a specific sequence.
There is a variety of software programs available that choose the most appropriate primers for a sequence such as the free, internet-based Primer3 (Rozen & Skaletsky, 2000) and Primaclade (Gadberry et al., 2005). There are a few general characteristics of primers that should be adhered to (Rybicki, 2001):
The primers should be between 17-28 nucleotide bases in length
The composition of the guanidine and cytosine (GC) bases should be 50-60%
The 3′ end of the primer should end in a guanidine or cytosine. Guanidine and cytosine are joined by three hydrogen bonds and are therefore stronger than the double hydrogen bonds of an adenine-thymine pairing.
The melting temperatures should be between 55-80°C
A series of three or more Gs or Cs at the 3′-ends of primers should be avoided.
As they are more stable, they may mis-prime at G or C-rich sequences
Complementary 3′-ends of primer pairs should be avoided. This may result in primer dimers forming
Primers that contain self-complementary regions will form secondary structure and be prevented from annealing
Previous workers have selected a range of sequences from different genome segments of the AHSV. The NS2 gene was used in the first PCR published to detect the AHS virus (1994). The NS2 gene has high sequence similarity within the
serogroup, but was divergent enough among serogroups not to detect other Orbiviruses. A single-tube RT-PCR was developed targeting regions on the VP7 and NS3 gene a year later and all nine serotypes were detected (Zientara et al., 1995b).
Following on from that work, the NS3 gene (Segment 10) was used to differentiate the nine serotypes using restriction fragment length polymorphism (RFLP). This novel method used restriction enzymes that hydrolysed the amplified regions resulting in specific, unique patterns on agarose gels (Zientara et al., 1995a). In 1997, VP7 was targeted again, coupled with a dot-blot hybridisation technique (Sailleau et al., 1997). In 2000, all nine serotypes were individually identified using 15 different primers in different combinations, but in nine separately optimised PCR runs (Sailleau et al., 2000). In South Africa, the first PCR assay was developed in 2004. It serotyped the virus using 16 primers under identical reaction conditions (Koekemoer & van Dijk, 2004). However, it required lengthy post-PCR analysis. Rodriguez-Sanchez (2008a) used the NS1 gene as a target and combined it with gel-based techniques. Most recently, the VP7 gene was used to develop an assay for AHS coupled with probe- based technologies (Fernández-Pinero et al., 2009).
2.9.6.3 cDNA synthesis
PCR can only work from DNA templates, as the polymerase used is a DNA polymerase. RNA therefore needs to be transcribed into DNA (complementary DNA or cDNA). When double stranded RNA is the initial nucleic material, such as is the case for the AHS virus, it must be denatured so that cDNA can be synthesised from it. This is achieved by either heat denaturation or adding a methyl mercuric hydroxide solution to dsRNA material (Wade-Evans et al., 1990; Zientara et al., 1995b) in the presence of each primer and incubating at room temperature for 10 minutes. Compared to heat denaturation, methyl mercuric hydroxide increases the sensitivity of RT-PCR by ten- fold (Wilson & Chase, 1993), although heat denaturation is the most often used in recent years. The now single stranded RNA can then be combined with a solution of each deoxynucleotide triphosphate (dNTP) (i.e. dATP, dTTP, dGTP and dCTP) in a suitable buffer and a reverse transcriptase. The solution will then undergo a series of temperature changes for example: incubation at 37°C for 1 hour, heated to 95°C for 5 minutes to denature the reverse transcriptase and chilled on ice for 5 minutes. The cDNA is stored at -20°C (Stone-Marschat et al., 1994).
2.9.6.4 PCR
The cDNA template can now be subjected to standard PCR protocols. One of the OIE recommended protocols is as follows: 30 cycles of 95°C for 1 minute, 42° for 1 minute, 70°C for 2 minutes and followed by 70°C for 10 minutes (Stone-Marschat et al., 1994).
Articles published recently have tended towards kit-based PCR protocols such as the Brilliant® QRT-PCR Master Mix One-Step kit (Rodriguez-Sanchez et al., 2008a), the Applied Biosystems GeneAmp Gold RNA PCR core kit (Quan et al., 2008) or the Qiagen One Step RT-PCR kit (Fernández-Pinero et al., 2009).
2.9.6.4.1 Real-time fluorogenic RT-PCR
To overcome the shortfalls of standard PCR, such as the gel-based nature of results and the increased time that this takes, Agüero et al. (2008) developed the first real-time fluorogenic RT-PCR for AHSV. This method was based on a TaqMan® probe and was directed towards Segment 7 of the AHSV genome. The authors claimed a 1000-fold increase in sensitivity compared to the OIE referenced method. TaqMan® probes are, unfortunately, not a cost-effective option. Hybridisation probes have also been used to serotype AHS isolates, and are an improvement on virus neutralisation assays (Koekemoer et al., 2000).
2.9.6.4.2 Nested RT-PCR
A nested RT-PCR has been developed with equal sensitivity to the real-time fluorogenic RT-PCR described above (Aradaib, 2009). Remarkably, the authors claim that the RT-PCR that they describe can detect as little as 0.1 fg of viral RNA, equivalent to six viral particles. In terms of a rapid, cost-effective assay, this assay was also successfully used directly on clinical samples (blood, lungs, liver, and spleen).
However, this method involves two PCR reactions that offset the time saved by the absence of RNA extraction procedures.