Detection Of Four Pathogens Of Emerging And Re-emerging Infectious Diseases With New Methods

SARS-associated coronavirus, Vibrio cholerae O139, Enterohaemorrhagic E. coli O157:H7, and bacillus anthracis are important pathogens of infectious diseases, which will be a severe threat to people's health and interfere with the development and cooperation of politics, economy and civilization. The rapid specific sensitive detection of these pathogens is very import for prevention and control of the epidemic of infectious diseases. In this paper, we describe the development of a double-antigen sandwich ELISA to detect antibodies to SARS-associated coronavirus in human serum, and the development of real-time PCR for quantitative detection of SARS-CoV, VCO139, EHEC O157:H7 and bacillus anthracis .A recombinant partial nucleocapsid protein of SARS-CoV screened out with protein array was applied as a serodiagnostic antigen in the ELISA. The cut-off OD of the ELISA was 0.15 . The intra-assay and inter-assay coefficients of variation (CV) were less than 5%. The specificity and sensitivity of the ELISA were demonstrated by using a panel set up by the Chinese National Institute for the Control of Pharmaceutical and Biological Products, including 18 positive and 20 negative sera and 1 serially diluted serum as a sensitivity control. All of 18 positive sera and none of 20 negative sera were shown positive, and the ELISA can detect the 64-fold dilution of sensitivity control serum with an OD of 0.162. The ELISA kit was tested with 3470 clinical serum samples, 70% of 442 SARS-confirmed and 76 % of 302 convalescent were detected positively, and only two of 2726 clinical samples from patients with other diseases or healthy individuals was shown weakly positive. These results indicate that the ELISA is effective as a screening method for serodianosis of SARS-associated coronavirus.In order to develop the real-time PCR for SARS-associated coronavirus, the primers and quantitative probes were designed and applied to detect coronavirus, based on the principle of complex probes quantitative assay. Sensitivity, specificity,reproducibility and range of quantitation of this method were determined, the quantitative RT-PCR for coronavirus with complex probes and an easy to handle and high efficiency method for isolation RNA from samples were established. The detect limit is 5 copies RNA per reaction and no negative samples or other RNA/DNA were detected with this method. It allows for a high sample throuput. It showes a very good precision, the coefficient of variation of threshold cycle was less than 5%. 42 clinical samples were detected with this quantitative RT-PCR. The rate of SARS samples can be detected was 79%. Itcan be concluded that the method established for quantitation of SARS-CoV is highly sensitive, rapid and easy to handle and shows a very good reproducibility, it can be applied to clinical diagnosis.A real-time PCR assay based on complex probe was developed to detect V. cholerae 0139. Targeted at the sequence encoding glycosyltransferases, primers and probes were designed and applied to detect 85 strains of V. cholerae 0139 and 78 strains of other enteric bacteria. All of the strains of V. cholerae 0139 tested are positively detected and none of the other bacteria was detected by the assay. The sensitivity of the assay was about lOOCFU/ml in pure cultures, and can positively detect lOcopies/ul of recombinant DNA. The correlation between Ct values and starting concentration (CFU/ml) of V. cholerae 0139 is linear and accurate quantitation can be achieved with samples detected within 101 - 107 CFU/ml. The reproducibility of the assay was also investigated, it showed the coefficient of variation of intra-assay and inter-assay was less than 5 %. The ability of this assay to detect food or environmental samples was assessed with spiked lake water. The seeded concentration of lOOCFU/ml can be detected by the assay. The real-time PCR was used to screen 335 diarrhea stool specimens. All the 90 V. cholerae O139 culture-positive and 43 V. cholerae O139 culture-negative stool specimens were positive by real-time PCR, and the remaining specimens were all negative by PCR. The results indicated that the real-time PCR assay is a reproducible, specific and sensitive method for the detection of V. cholerae 0139 in food or environmental samples.The development of a real-time PCR assay to detect the prescence of EHEC O157:H7 using complex probe was described. Complex probes were designed to recognize the rfbE gene, coding for an enzyme necessary for O-antigen biosynthesis. The specificity of the complex probe-based PCR assay was evaluated using 13 strains EHEC O157:H7 and 78 strains of other enteric bacteria. All O157:H7 tested was positively identified while all other species were not detected by the assay. Posititve detection of EHEC O157:H7 was demonstrated when only 1 CFU of bacteria and 1 copy of plasmid DNA in the samples. The correlation between Ct values and starting concentration (CFU/ml) of EHEC O157:H7 is linear and accurate quantitation can be achieved with samples detected within 10° - 106CFU/ul. The reproducibility of the assay was also investigated, it showed the coefficient of variation of intra-assay and inter-assay was less than 5 %. The capability of the assay to detect EHEC O157:H7 in raw milk , orange juice and soil was demonstrated. 1 X 103 CFU/ml in spiked raw milk and orange juice and 1 X 103 CFU/ml /O.lg in spiked soil was detected.. The results indicated that the real-time PCR assay is a reproducible, specific and sensitive method for the detection of EHEC O157:H7 in food or environmental samples.Quantitative analysis of anthrax spores from environmental samples is essential for accurate...