Molecular Diagnostics

• Submission Guidelines
• Molecular Virological Assays
  • PCR
• Molecular Assays for Differentiation and Genetic Characterization

Molecular Virological Assays

In general, samples appropriate for conventional microbiological evaluation (i.e., detection of viruses) are also suitable for molecular diagnostic tests. This includes excretions and secretions, feces, blood, serum, and biopsies from infected, live animals (i.e., ante mortem), and relevant tissues/organs and washings (e.g., bronchial lavage) from necropsied animals (i.e., post mortem). Refer to the Sample Collection and Submission Guidelines for comments on sample handling and shipment.

For best results, clinical specimens should be collected aseptically from individual animals. Care should be taken to avoid cross contamination. Samples from different animals can be pooled for molecular assays if cost is a concern, but it is recommended that clients consult with VDL staff prior to pooling samples. Alternatively, samples can be pooled in the lab.

Several molecular diagnostic methods are available for detection or differentiation of viruses, including polymerase chain reaction (PCR), in situ hybridization (ISH), fingerprinting, and sequencing. Some, but not all, molecular techniques first require isolation of the target agent.

Polymerase chain reaction (PCR)

PCR is a process by which a portion of viral nucleic acid (DNA or RNA) is replicated a million times or more. Detection of this amplified product (amplicon) indicates that the sample is positive for the target virus.

Proper amplification relies on a set of two short synthetic oligonucleotides (primers). Good test performance requires that primers bind only to the corresponding nucleotide sequences of the viral genome and nothing else. Thus, the specificity of the assay and accuracy of the results depend upon the design of PCR primers. Theoretically, a PCR-based assay is capable of detecting one copy of the viral genome. However, depending upon the target agent, type of sample, and condition of sample, diagnostic sensitivity often is not as sensitive as anticipated and, depending on the circumstances, conventional assays may provide equivalent test performance. Also, PCR is expensive relative to most other diagnostic tests and positive results do not always have biological significance, i.e., PCR reacts with inactivated viruses, as well as infectious viruses.

There are several types of PCR assays:

• RT-PCR is used to detect target RNA from clinical specimens. Reverse transcription (RT) of RNA is required to make complementary DNA for further amplification. This assay is most frequently used for specific detection of RNA viruses.
• Nested PCR is a PCR done in two steps, a primary PCR reaction and a nested reaction. The primary (or first) reaction uses a set of primers to generate a product that serves as the template for the nested (or second) reaction. The nested reaction uses a set of PCR primers specific for a region within the amplified product from the first reaction. Therefore, the nested reaction often serves as a confirmation for the specificity of the PCR products amplified in the primary reaction.
• Multiplex PCR is a PCR designed to detect more than one target sequence in a single PCR reaction. The assay uses two or more sets of primers. Each set of primers is specific for a different target sequence. The assay is most commonly used for simultaneous detection of multiple viral genes and differentiation of genotypes or subtypes of related microorganisms.
• Real-time PCR combines PCR amplification and detection into a single step. The basic principle of real-time quantitative PCR is the detection of target sequences using a fluorogenic 5' nuclease assay (often called "TaqMan"). The advantages of this system include high reproducibility, the capability of handling large numbers of samples, the potential for quantitative results, and decreased turnaround time. The disadvantages include high instrument cost and the requirement for technical proficiency.

Molecular Assays for Differentiation and Genetic Characterization

Differential PCR can sometimes be used to distinguish closely related targets. Differential PCR is done either in a multiplex format using two or more sets of primers or by running two separate PCR assays. Refer to the Table of Molecular Assays for a list of differential PCRs.

RFLP analysis is a molecular differential technique that, to a limited extent, can distinguish between two viruses at the genomic level. RFLP is based on the fact that restriction enzymes recognize specific nucleotide sequences and cut the genome at that location. In general, the RFLP procedure consists of isolating the target microorganism from a clinical specimen, extracting DNA or RNA, digesting the nucleic acid material with restriction enzymes, and gel electrophoresis of the resulting products. The pattern of fragments observed on the gel is used to characterize or compare isolates. In general, RFLP requires virus isolates; however, RFLP using PCR products instead of native nucleic acid has recently been developed. This method provides faster turnaround since it is not necessary to isolate and propagate virus.

RFLP is useful for differentiating minor differences at the strain level that normally cannot be detected by any antigenic assays, such as tissue immunoassays, serology, and enzyme immunoassays. RFLP analysis is rapid and less expensive than sequence analysis, but RFLP will miss many of the genetic differences that are revealed by sequencing.

Sequence analysis is a molecular tool for use in characterizing the genetic information of microorganism in detail. Sequencing provides a list of the nucleotides in the viral genome in the order in which they appear. Comparison of the genetic information makes complete differentiation between viruses possible. Either partial or whole genomic sequencing can be done, depending upon the size of genome and purpose of the analysis. In addition, RFLP patterns and amino acid composition can be predicted from sequence data. One disadvantage of sequencing is the expense of conducting the analysis.