This application relates generally to systems and methods for analyzing a sample for the presence of one or more nucleic acids under closed conditions and, more particularly, to a cap for a vessel used for performing such analyses, especially nucleic acid amplification reactions such as polymerase chain reaction (PCR).
Nucleic acid amplification reactions are crucial for many research, medical, and industrial applications. Such reactions are used in clinical and biological research, detection and monitoring of infectious diseases, detection of mutations, detection of cancer markers, environmental monitoring, genetic identification, detection of pathogens in biodefense applications, and the like, e.g., Schweitzer et al., Current Opinion in Biotechnology, 12: 21-27 (2001); Koch, Nature Reviews Drug Discovery, 3: 749-761 (2004). In particular, polymerase chain reactions (PCRs) have found applications in all of these areas, including applications for viral and bacterial detection, viral load monitoring, detection of rare and/or difficult-to-culture pathogens, rapid detection of bio-terror threats, detection of minimal residual disease in cancer patients, food pathogen testing, blood supply screening, and the like, e.g., Mackay, Clin. Microbiol. Infect., 10: 190-212 (2004); Bernard et al., Clinical Chemistry, 48: 1178-1185 (2002). In regard to PCR, key reasons for such widespread use are its speed and ease of use (typically performed within a few hours using standardized kits and relatively simple and low cost instruments), its sensitivity (often a few tens of copies of a target sequence in a sample can be detected), and its robustness (poor quality samples or preserved samples, such as forensic samples or fixed tissue samples are readily analyzed), Strachan and Read, Human Molecular Genetics 2 (John Wiley & Sons, New York, 1999).
Despite the advances in nucleic acid amplification techniques that are reflected in such widespread applications, there is still a need for further improvements in speed and sensitivity, particularly in such areas as infectious disease detection, minimum residual disease detection, bio-defense applications, and the like.
Significant improvements in sensitivity of PCRs have been obtained by using nested sets of primers in a two-stage amplification reaction, e.g., Albert et al., J. Clin. Microbiol., 28: 1560-1564 (1990). In this approach, the amplicon of a first amplification reaction becomes the sample for a second amplification reaction using a new set of primers, at least one of which binds to an interior location of the first amplicon. While increasing sensitivity, the approach suffers from increased reagent handling and increased risk of introducing contaminating sequences, which can lead to false positives.
Significant improvements in sensitivity and a reduction of false positives have also been obtained by carrying out reactions in closed environments. A drawback of highly sensitive amplification techniques is the occurrence of false-positive test results, caused by inappropriate amplification of non-target sequences, e.g., Borst et al., Eur. J. Clin. Microbiol. Infect. Dis., 23: 289-299 (2004). The presence of non-target sequences may be due to lack of specificity in the reaction, or to contamination from prior reactions (i.e. “carry over” contamination) or to contamination from the immediate environment, e.g., water, disposables, reagents, etc. Such problems can be ameliorated by carrying out amplifications in closed vessels, so that once a sample and reagents are added and the vessel sealed, no further handling of reactants or products takes place. Such operations have been made possible largely by the advent of “real-time” amplifications that employ labels that continuously report the amount of a product in a reaction mixture.
Some processes such as nested PCR involve two processes performed in sequence. For instance, a conventional nested PCR procedure utilizes two sequential amplification processes, which include a first round reaction for amplifying an extended target sequence with outer primers, and a second round reaction for amplifying an internal sequence from the product of the first round reaction with inner primers. The internal sequence may or may not overlap with one of the ends of the extended sequence. The enhanced sensitivity of the nested PCR is achieved by carefully controlling the reaction conditions for the first and second amplification processes to favor the generation of the desired product. Unfortunately, the high sensitivity provided by the nested PCR procedures is achieved at the price of potential false positives as the reaction tubes containing high concentrations of the first amplicons have to be opened and manipulated to set up the second amplification, thereby introducing the chance of contamination, which is a significant cause of false-positive results and diminishes the reliability of the analysis.