Discussion

We have demonstrated that our simple and reproducible AluI RFLP assay in combination with allele specific PCR can be successfully used for the accurate quantitation of the relative amount of CRM197 tox gene allele in bacterial DNA samples，as well as for detection of greater than 5%wild-type contamination.

In addition to the RFLP technique，there are several alternative approaches that are currently being used for diagnostics of point mutations，and these could potentially be applied in CRM197 diagnostics.However，these alternative approaches have certain disadvantages when compared to RFLP.Some of those，such as allele-specific oligonucleotide hybridization，oligonucleotide ligation assay，and direct sequencing，appear to be more laborious than our assay，and often require radioactive labeling，which presents safety problems.Others，such as single strand conformational polymorphism，denaturing gradient gel electrophoresis，and mismatch cleavage，have certain limitations with respect to particular bases or mismatches［15-17］.

The most popular approach used so far for estimation of allele concentration in DNA samples is quantitative PCR with allele-specific primers［18］.However，the reliability of quantitation with this procedure may be significantly affected by unequal efficiency of amplification with different allele-specific primers.In contrast，our Alu I RFLP quantitation assay uses a pair of primers that are not allele-specific.Therefore，both CRM197 and non-CRM197 allele templates are amplified at random，and with equal efficiency.As a result，the initial allele ratio in the template DNA is correctly represented in the PCR product，and this is the basic advantage of the quantitative RFLP design.

Figure 4　PCR product patterns with CRM197+wild-type DNA mixtures in template diagnostics by allele-specific PCR（2%agarose gel.Lane 1—Lane 7∶100%，99%，95%，90%，50%，10%，and 0%of CRM197-Ref.in the template mixture.Lane 8：Molecular weight marker XIV（Boehringer Mannheim）

We have complemented the AluI RFLP assay by an allele-specific PCR assay.Each assay has different advantages，and both work well in combination.The AluI RFLP assay is quantitative，while the allele-specific PCR is not.On the other hand，the allele-specific PCR assay can differentiate both wild-type and CRM197 tox gene alleles，while the AluI RFLP assay is specific for CRM197 allele only.Both assays have same sensitivity，i.e.，both are able to detect a minimum of 5%particular allele in the template sample.

The PCR-RFLP procedure described in this article is the first DNA assay applied to the CRM197 toxin protein.So far，the toxicity of CRM197 has been evaluated by using the lethality test in guinea pigs，the cytotoxic activity assay，and the ADP-ribosyltransferase enzymatic assay［19］.We believe that our PCR-RFLP procedure could become an important initial control step for estimation of the genetic purity of bacterial culture prior to manufacturing of CRM197 protein.However，the final protein toxicity assays are also required to ensure vaccine safety.