Braun LJ. 1999;1:37-40 Diagnostic porcine polymerase chain react

Diagnostic notes	January and February, 1999

Diagnostic porcine polymerase chain reaction assay: The future is here

Lyle J. Braun; Christopher C.L. Chase, DVM, PhD

LJB, CCLC: Animal Disease Research and Diagnostic Laboratory; Veterinary Science Department, South Dakota State University, Brookings, South Dakota 57007

This diagnostic note has been refereed.

This is the first in a series of three articles describing diagnostic techniques.

Braun LJ, Chase CCL. Diagnostic porcine polymerase chain reaction assay: The future is here. Swine Health Prod.1999;7(1):37-40.

This article available in PDF format. (184k)

Summary

This report discusses the relative importance of polymerase chain reaction to veterinary diagnostics. A brief description of the assay procedure is included.

Keywords: polymerase chain reaction, diagnostics

Received: July 6, 1998
Accepted: October 19, 1998

To devise proper treatment and prevention programs, one must be able to accurately detect and identify porcine pathogens. One should judge the relative merit of the many diagnostic tools available to accomplish this on the basis of the "three Ss"–speed, sensitivity, and specificity. Polymerase chain reaction (PCR) is a new assay that promises improvement in all three Ss^2,3 over the most commonly used diagnostic methods.

In terms of the three Ss, serology and isolation have some inherent limitations:

• they can be slow–it can take days to months to obtain results;
• the sensitivity of these methods is relatively low;
• they can only be used with a limited sample range (tissue, feces, or blood);
• they require that the samples be fresh; and
• they cannot detect some organisms, such as swine hepatitis E and papilloma virus.¹

Polymerase chain reaction is the wave of the future in diagnostics, because it increases sensitivity (it can detect as few as 10 organisms in a sample), specificity, and speed (it can be completed within several hours). Any type of sample can be used with PCR, which identifies pathogen (virus, bacteria, or parasite) and host genes. Thus, the PCR assay allows us to detect pathogenic organisms that may not have been detectable using other methods.

Some critics of PCR consider the technique too sensitive, because PCR detects the pathogen’s genetic material whether the pathogen is dead or alive. Polymerase chain reaction can result in false positives from vaccine strains, contaminates, normal flora, or nonviable organisms. The increased specificity of PCR, however, can also be exploited to differentiate vaccine strains from field strains. This specificity is achieved with primers–short fragments of synthetic DNA that are 18-25 bases long. These primers are designed to match a unique region of the target DNA. If successfully designed, the assay can differentiate a field strain from a vaccine strain. In studies with porcine reproductive and respiratory syndrome virus (PRRSV), vaccine-specific primers detected the vaccine strain months after the initial vaccination.⁴

Sampling

Many different sample types can be used for the PCR assay–serum, whole blood, urine, semen, and tissues. The optimal sample will depend on the organism of interest and the specific PCR assay implemented. To reduce the possibility of cross contamination, specimens should be collected aseptically, with single-use devices. The fresh specimens should be shipped on ice for next day delivery; however, certain specimen types may be frozen or formalin-fixed prior to submission. Always contact the diagnostic laboratory to determine how to collect and ship the sample.

The PCR assay

PCR is used to amplify a specific DNA fragment that is between 150-1000 base pairs long. This DNA fragment is called the "target sequence." After the sample arrives at the diagnostic laboratory, the nucleic acid is first extracted. Extraction, the first step for all specimen types, removes excess protein and inhibitory substances, leaving pure, full-length DNA or RNA for the PCR assay.

Since the starting nucleic acid must be DNA, the isolated DNA from bacteria, parasites, or DNA viruses can be used directly. RNA from RNA viruses must first undergo reverse transcription (RT), an enzymatic process that converts the RNA into cDNA (a DNA strand complementary to the RNA), allowing the target sequence to be amplified (Figure 1).

The PCR assay itself is divided in three distinct stages:

• Denaturation. Occurs when DNA is heated to 95 degrees C (Figure 2a). This causes the double-stranded DNA to "unzip," creating two single strands of DNA.
• Annealing. The temperature is reduced to 50 degrees C to allow the primers to anneal or bind to the single-stranded DNA target (Figure 2b). Properly designed primers will bind only to the target region of the sequence, yielding an assay with high specificity.
• Elongation. The target sequence, primers, free-dNTPs, and Taq polymerase are heated to 72 degrees C for the elongation step (Figure 2c). In elongation, the free-dNTPs are paired up with the target sequence and cemented together by the Taq DNA polymerase to form a new double-stranded DNA molecule.

These three steps constitute one cycle of amplification. Denaturation, annealing, and elongation are repeated over and over on the automated thermal cycler to amplify the DNA. After 30-40 cycles (2-3 hours), the initial DNA has been geometrically amplified (Figure 2c), providing billions of DNA copies (PCR product) from just one copy of target DNA. The geometric amplification is the sensitivity component of the assay.

Gel electrophoresis

The amplified product is then detected by agarose gel electrophoresis. Gel electrophoresis is a technique by which one can sort DNA fragments of different sizes. Electric current is passed through the gel, which causes the DNA to migrate. Shorter fragments will travel farther than longer fragments.

Fragments of known size are placed into the first lane and a fraction of the PCR product is placed into consecutive lanes. Then, current is applied and the DNA is allowed to migrate. The gel is stained with a fluorescent dye (ethidium bromide) which will only bind to the DNA present in the agarose gel. The gel is visualized with an ultraviolet light and documented in a photograph (Figure 3). The size of the amplified PCR product is determined and compared to standard controls. Other detection techniques are being developed that will result in a colormetric reaction, reducing the time needed to complete the assay and allowing quantification of the amount of PCR product.

Currently, there are PCR assays for both porcine viral and bacterial agents. Assays are currently available to detect the following viruses:

• PRRSV,⁵
• transmissible gastroenteritis virus (TGEV),⁶
• porcine respiratory coronavirus (PCRV),⁷ and
• pseudorabies virus (PRV).⁸

Some of the bacterial pathogens that can be detected by PCR include:

• Lawsonia intracellularis,
• Serpulina hyodysenteriae,
• Salmonella spp, and
• Mycoplasma spp.^9,10

Many more PCR assays are sure to be made available for pathogenic organisms and their subspecies whose importance warrants the development of specific assays.

The improvement in the three Ss–sensitivity, specificity, and speed–offered by PCR have made it an invaluable tool in diagnostic laboratories. Although PCR offers many benefits, it is important to use it appropriately. PCR is not recommended, for example, as a herd screening tool because of its expense. Its use should be reserved for situations in which it is essential to know whether a specific pathogen is present–e.g., boar testing or testing for persistently infected animals.¹¹ As with other technological advances, PCR testing will become more affordable and routine as its use evolves.

References

1. Meng, et al. A novel virus in swine is closely related to the human hepatitis E virus. PNAS. 1997; 94: 9860-9865.

2. Suarez P, Zardoya R, Pricto C, Solana A, Tabares E, Bautista JM, Castro JM. Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR). Arch Virol. 1994; 135:89-99.

3. Van Woensel P, Van Der Wouw J, Visser N. Detection of porcine reproductive respiratory syndrome virus by the polymerase chain reaction. J Virol Methods. 1994; 47: 273-278.

4. Wesley RD, Mengeling WL, Andreyev V, Lager KM. Differentiation of vaccine (strain RespPRRS) and field strains of porcine reproductive and respiratory syndrome virus by restriction enzyme analysis. Proc AASP Ann Meet. 1996; 141-143.

5. Christopher-Hennings J, Nelson EA, Nelson JK, Hines RJ, Swenson SL, Hill HT, Zimmerman JJ, Katz JB, Yaeger MJ, Chase CCL, Benfield DA. Detection of porcine reproductive and respiratory syndrome virus in boar semen by PCR. J Clin Microbiol. 1995; 33(7): 1730-1734.

6. Woods RD. Development of PCR-based techniques to identify porcine transmissible gastroenteritis coronavirus isolates. Can J Vet Res. 1997; 61:167-172.

7. Paton D, Ibata G, Sands J, McGoldrick A. Detection of transmissible gastroenteritis virus by RT-PCR and differentiation from porcine respiratory coronavirus. J Virol Methods. 1997: 66: 303-309.

8. Maes RK, Beisel CE, Spatz SJ, Thacker BJ. Polymerase chain reaction amplification of pseudorabies virus DNA from acutely and latently infected cells. Vet Microbiol. 1994;24:281-295.

9. Elder RO, Duhamel GE, Mathiesen MR, Erickson ED, Gebhart CJ, Oberst RD. Multiplex polymerase chain reaction for simultaneous detection of Lawsonia intracellularis, Serpulina hyodysenteriae, and salmonellae in porcine intestinal specimens. J Vet Diagn Invest. 1997; 9(3): 281-286.

10. Smith SH, McOrist S. Development of persistent intestinal infection and excretion of Lawsonia intracellularis by piglets. Res Vet Sci. 1997; 62:6-10.

11. Christopher-Hennings J, Nelson EA, Hines RJ, Nelson JK, Swenson SL, Zimmerman JJ, Chase CCL, Yaeger MJ, Benfield DA. Persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars. J Vet Diagn Invest. 1995;7:456-464.

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