Development Of A Quantitative PCR Method Using A Live Bacterium As Internal Control For The Detection Of Listeria Monocytogenes

Listeria monocytogenes is an important food-borne pathogen that causes listeriosis. It is widely distributed in the environment, and its infections can be life threatening, with a high fatality rate of 20 to 30%. It was estimated that approximately 2,500 individuals have developed listeriosis in the United States per year and the costs of the acute illness from foodborne Listeria are $2.3 billion. At the same time, L. monocytogenes has emerged as a significant foodborne pathogen in China during the past few years, along with a trend toward increasing consumption of ready-to-eat food products. Therefore, rapid detection methods are of significant importance to the L. monocytogenes quality control programs, which have to be applied throughout the food production chain.In order to overcome the limitations of traditional PCR method, a real-time quantitative PCR (Q-PCR) assay was developed with a mutant strain used as an internal control. This technique is suitable for most existing real-time PCR protocols, which can be used to quantify the initial amounts of L. monocytogenes in enriched samples and monitor the false negative results during the whole process. The main results are as follows:1. Four different bacterial detection methods (BIOLOG, VITEK, CRYSTAL and PCR) have been evaluated by L. monocytogenes CMCC 54002. The results showed that the PCR method is the best among these detection methods. The other three methods are time-consuming during sample preparation and request for separating the individual colonies from agar plates. Moreover, they can't distinguish between L. monocytogenes and other Listeria species. In contrast, the PCR method can identify L. monocytogenes by specific primers and doesn't need to purify the samples. Further serological analysis showed that the nine L. monocytogenes strains used in this study are serotypes 1/2a, 4b, 1/2c, 3b which are the main serotypes in most listeriosis cases.2. An IAC design method was developed by the computational DNA random shuffling. The hly gene was selected as a target gene for detecting L. monocytogenes by Q-PCR, and the conserved regions of the hly gene were used to design suitable L. monocytogenes-specific PCR primers and probe. Then, a DNA random shuffling software was used to shuffle the probe binding sequence. A total of 1000 random shuffling sequences were generated. Based on results from Bacon Designer 5.0 software analyses and BLAST-N searches, an IAC fragment that did not display significant similarity to any known pathogen DNA sequence was identified and synthesized by using the overlap-extension PCR technology. The IAC and hly amplicon share the equal length (65 bp) and GC content (55.4%), which ensures the equal PCR amplification efficiencies.3. A mutant L. monocytogenes (LM-IAC) was obtained by exchanging the chromosomal hly gene with the IAC fragment through homologous recombination technology. The flanking regions of the hly gene, an IAC fragment and a kanamycin resistance gene were inserted into the pKSV7 shuttling vector, which contains a temperature sensitive replicon, to generate a recombinant vector called pKSV7-UIKD. A restoring strain was obtained by the replica plating method after continuous passages in BHI broth medium. Bacteria in colonies which could not grow on the chloramphenicol resistant plates, but can grow on the kanamycin resistant plates were candidates for the restoring strain. The recombination rate was about 1.3% (4/300). The restoring strain was identified by PCR, RT-PCR and sequencing analysis to further confirmed that the IAC fragment and the kanamycin resistance gene had replaced the hly gene on the restoring strain chromosome.4. A LM-IAC AQ-PCR assay was developed for rapid and accurate detection of the original amount of wild-type L. monocytogenes before DNA isolation. (1) The amplification efficiencies and template extraction efficiency were estimated. The calculated amplification efficiencies of the hly amplicon and IAC were 1.05 and 1.06, respectively. The calculated template extraction efficiency results was similar (P > 0. 05) between wild-type L. monocytogenes and LM-IAC (about 10%) over a five orders of magnitude. (2) A calibration curve was established with the log (X/R) (X is the initial number of template, R is the initial number of bIAC) plotted againstΔC_T (C_TLM -C_TLM-IAC). The calibration curve calculated by linear regression was y = -0.313x - 0.0773 with a square regression coefficient of 0.9997. Comparison of the quantified wild-type L. monocytogenes contents with known amounts indicated that the established LM-IAC Q-PCR system was more reliable than the traditional absolute quantification method.5. A LM-IAC EQ-PCR assay was developed for monitoring the false negative results in the whole control. (1) The biochemical characteristics between wild-type L. monocytogenes and LM-bIAC have been compared with BIOLOG and VITEK's identification system. The results showed that the two strains have similar biochemical characteristics. (2) The uniform design experiments were performed to analyze the comprehensive effects of different LM-IAC contents in medium on wild-type L. monocytogenes growth. It was found that the wild-type L. monocytogenes growth was stable with 10² cfu -10⁴ cfu LM-IAC contents. The generation time of wild-type L. monocytogenes and LM-IAC were 49 min and 55 min respectively. (3) Milk samples, chiken samples and pickle meat samples were artificially contaminated about 1000 cfu wild-type L. monocytogenes and LM-IAC and incubated with UVM selective enrichment broth, at the same time, preparing ddH₂O as positive control. Wild-type L. monocytogenes and LM-IAC which contaminated the ddH₂O and milk samples could be detected successfully by the LM-IAC Q-PCR assay without enrichment. However, wild-type L. monocytogenes and LM-IAC which contaminated the chicken and pickle meat samples could be detected after 3 h enrichment and 6 h enrichment respectively. It was demonstrated that the LM-IAC EQ-PCR assay could successfully eliminate the false-negative results between chicken and pickle meat samples. Therefore the advanteges of the LM-IAC EQ-PCR assay are accuracy and time- saving.6. A single multiple internal amplification control (mIAC) DNA molecule was constructed to detect the L. monocytogenes, Salmonella and Vibrio parahaemolyticus. Three pairs of primers (hlyAF/AR, invAF/ invar, toxRF/ toxRR) were designed based on the species-specific sequences of hlyA gene, invA gene and toxR gene respectively. The mIAC was synthesized by the overlap-extension PCR technology which contains the three pairs of primers' sequences. Sensitivity of those mIAC PCR systems for purified target DNA were 73. 0 fg/μl (L. monocytogenes), 5.04 fg/μl (Salmonella) and 76. 4 fg/μl (V. parahaemolyticus) respectively. In order to estimate the mIAC, 40 chiken samples, 60 milk samples and 60 shrimp samples were artificially contaminated with L. monocytogenes, Salmonella and Vibrio parahaemolyticus respectively. After PCR amplification, 3 chicken samples, 2 milk samples and 3 shrimp samples which turned out to be false-negative. The results demonstrated that the systems with mIAC could successfully eliminate the false-negative results.