Controls

Procedural controls for M-PCR are similar to controls that should be used for conventional PCR. These consist of both positive and negative controls, which are designed to monitor the sensitivity and specificity of each amplification run, respectively. The preparation of a batch of clinical specimens to be tested by PCR should include up to six negative specimens, depending on the number of specimens being tested. Use two negative controls for a batch of six specimens and six for a batch of 20...

Transfer of DNA Fragments to Membranes

Transfer of DNA fragments to a membrane, as described by Southern, was achieved using nitrocellulose and upward capillary transfer. Since 1975, several modifications have been made that simplify, accelerate, and increase the sensitivity of blotting. The most significant improvement has been in the immobilizing membrane itself. Although nitrocellulose is still used in many laboratories, nylon membranes have several advantages including greater tensile strength, superior nucleic acid binding...

Difficulties And Disadvantages

Although researchers have been using molecular diagnostic methods to detect viral nucleic acids in clinical specimens for nearly 30 years, the transition from the research laboratory to the clinical laboratory has been slow and painful. Early nucleic acid hybridization tests were more expensive and more labor intensive, and had unacceptably long turnaround times compared with existing antibody methods. In addition, these tests often used radiolabeled probes, which were disadvantageous in the...

ADNA Hybridization Assay Formats

Several DNA hybridization assay formats including membrane-based, solution, and in situ hybridization have been coupled with chemiluminescence for the detection of viruses and other infectious agents. Membrane-based chemiluminescent hybridization assays have employed either 1,2-dioxetane substrates for alkaline phosphatase or the enhanced chemiluminescence reaction of luminol and HRP, and are imaged on X-ray or photographic films or imaged directly and quantified using a CCD camera system....

Amplicon Analysis

Gel electrophoresis followed by hybridization with labeled DNA probes has been used to verify specific amplification products. If primer pairs are chosen so amplicons differ in size by 50-100 bp, agarose gel electrophoresis can be used to resolve M-PCR amplicons. Figure 1 shows the separation and identification of a 390-bp amplicon for N. gonorrhoeae and a 241-bp amplicon of C. trachomatis produced by M-PCR. With a well-characterized M-PCR assay that gives few or no nonspecific amplification...

Chemiluminescence Detection Systems

Alkaline phosphatase-dioxetane chemiluminescence systems have been used in a wide variety of DNA hybridization assays for detection of infectious Selected Studies That Have Used Chemiiuminescence to Detect Viruses, Chlamydia trachomatis, and Other Microorganisms Feline infectious peritonitis virus Grapevine closterovirus Hepatitis B virus Hepatitis C virus Herpes simplex virus Musiani et al. (1991a,1992) (DX) Yang et al. (1991) (DX) Yang et al. (1991) (DX) Vlieger et al. (1992) (LU) Bronstein...

Strengths and Weaknesses

The obvious advantage of M-PCR is the ability to detect more than one agent in a single test. For specimens such as respiratory tract secretions, from which several different viruses can be recovered, this ability offers potential cost savings. The second advantage of M-PCR is its high degree of sensitivity and ability to detect both noncultivatable virus and neutralized virus present in antigen-antibody complexes. As technology advances, target quantification, which is now working its way into...

Potential Applications

In his 1988 review, Tenover stated that the goal of DNA probe technology was to eliminate the need for routine viral, bacterial, and fungal cultures Tenover, 1988 . Although this goal could eventually be reached, the principal advantage of molecular diagnostic methods in current clinical virology laboratories is in the detection of nonculturable agents such as human papilloma virus, human parvovirus, astroviruses, caliciviruses, hepatitis B virus, and hepatitis C virus. Molecular methods are...

Components Of The bDna Assay

The bDNA assay differs from many other amplification procedures because the components are all supplied by the manufacturer Chiron Corporation . Therefore, this chapter describes the assay and its components but not their synthesis, since synthesis of branched nucleic acids would be impractical in a virology laboratory. Specific instructions and troubleshooting guidelines are provided in package inserts and in training sessions. The bDNA assay consists of target probes that mediate capture,...