Detecting and identifying bacteria is important in various medical and public health contexts and is important in controlling the quality of the food chain. Innumerable protocols, proprietary apparatus and kits have evolved to meet the needs of the rapidly growing field of bacterial detection. These require highly trained and skilled personnel to carry out the necessary procedures and even more highly trained and skilled personnel to evaluate the results. Moreover, many of the existing tests are extremely sensitive to environmental factors, such as growth and storage conditions and the presence of and competition from other bacteria or microorganisms. This puts an even greater emphasis on the need for exacting procedures and highly skilled operatives. Tests of the prior art are often expensive both with regard to reagents and to the apparatus in which the tests are run. Additionally, these tests require confirmation since they often are not adequately selective or inclusive resulting in both false positive and false negative results.
Polymerase chain reaction (PCR) is a powerful analytical tool permitting the amplification of any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof. The use of this procedure for bacterial detection has been reported in the literature. However, as it is commonly practiced, it is a procedure which demands adherence to strict protocols under strict conditions and requires personnel of advanced skills and training in order to achieve a reliable result.
In all DNA based methods for detection of organisms, and particularly in the PCR test procedure, extraneous components that may enhance or inhibit the test reaction make obtaining creditable results difficult. This occurs in testing food-derived matrices for bacterial contaminants that effect quality such as pathogens, spoilage, and off-taste promoters and the like. Because the test results in such circumstances are critical, it is important to evaluate the effectiveness of any particular test. The invention provides a positive control which is useful in establishing test validity.
The test procedure of the invention is PCR-based. In U.S. Pat. No. 4,683,202, basic to that art, Mullis describes a procedure in which separate, complementary-strands of the nucleic acid are treated with a molar excess of two oligonucleotide primers and the primers are extended to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The steps of the reaction, a sequence of thermal treatments, are carried out stepwise or simultaneously and are repeated as often as desired. Typically as many as thirty-five or more cycles are necessary to obtain a number of replicas adequate for further processing.
In U.S. Pat. No. 4,683,195, Mullis et al. teach that a specific nucleic acid sequence may be cloned into a vector by using primers to amplify the sequence which contain restriction sites on their non-complementary ends and a nucleic acid fragment may be prepared from an existing shorter fragment using the amplification process.
PCR has several applications designed to detect the presence of a specific DNA sequence only by amplification. It would be extremely advantageous to include a control reaction in any such PCR test because, when a test is negative for a target, it is important to know if that result is true or if the reaction failed due to instrument malfunction or inhibition of the reaction due to sample matrix effects. The latter is particularly common in testing food samples to determine the presence of pathogens or other organisms harmful to product quality. Samples containing, for example, cocoa, a potent inhibitor of PCR, may well contain a pathogen that will be masked in an uncontrolled PCR-based test. A positive control replication composition and method is the subject of the instant invention.
Williams et al. in "DNA Polymorphisms Amplified by Arbitrary Primers are Useful as Genetic Markers", Nucleic Acid Research, Vol. 18, No. 22, p. 6531-6535 and Welsh et al., "Fingerprinting Genomes using PCR with Arbitrary Primers", Nucleic Acid Research, Vol. 18, No. 22, p. 7213-7218 both demonstrate the use of single, arbitrary primers in a DNA amplification reaction to generate a characteristic pattern of amplification products from genomic DNA from a variety of sources including bacteria. In WO93/11264, Jensen et al. teach the use of a single arbitrary primer across a broad spectrum of microorganisms. Control reactions are not addressed.
Shuldiner et al., in PB92-100932 NTIS, teach detecting an RNA sequence by tagging the sequence with a unique random nucleotide sequence during reverse transcription. The unique nucleotide sequence is then utilized to selectively amplify the resulting DNA sequence reducing the number of false positives obtained as a result of contaminating DNA such as from an endogenous source or from carry-over. This procedure lacks the control aspects of the instant invention which permit avoiding false negatives as well as false positives.
Tercero et al. (EP 586112), teach a vector useful as positive control in PCR amplification. The vector contains a sequence substantially identical to that of a primer used in the procedure which, after amplification, yields a product differing in size from that produced by the target. If only the vector is amplified the result is a true negative, but if neither vector nor target are amplified then the test must be faulty. Requisite in such a control protocol is some means to separate the different size products. Because the control and the target reactions are carried out in the same vessel and co-amplified, there are competing reactions that, in some circumstances reduce the sensitivity of the procedure. This results from preferential amplification of one of the targets. Also required are reference for size of product DNAs since in the case of only a single amplification product it must be determined whether it is test product or control product. Thus, the disclosure of Tercero et al. does not address homogeneous detection and is not adapted thereto.
In summary, the literature does not disclose a bacterial test method that 1) uses simplified molecular biology techniques that require no special skills in preparing and handling reagents and in carrying out the protocol, 2) is insensitive to environmental factors affecting phenotypic expression, and 3) is both selective and inclusive and has a positive control integrated into the protocol.