Microarray analysis of Escherichia coli O157:H7.

AIM: To establish the rapid, specific, and sensitive method for detecting O157:H7 with DNA microchips. METHODS: Specific oligonucleotide probes (26-28 nt) of bacterial antigenic and virulent genes of E. coli O157:H7 and other related pathogen genes were pre-synthesized and immobilized on a solid support to make microchips. The four genes encoding O157 somatic antigen (rfbE), H7 flagellar antigen (fliC) and toxins (SLT1, SLT2) were monitored by multiplex PCR with four pairs of specific primers. Fluorescence-Cy3 labeled samples for hybridization were generated by PCR with Cy3-labeled single prime. Hybridization was performed for 60 min at 45 degrees. Microchip images were taken using a confocal fluorescent scanner. RESULTS: Twelve different bacterial strains were detected with various combinations of four virulent genes. All the O157:H7 strains yielded positive results by multiplex PCR. The size of the PCR products generated with these primers varied from 210 to 678 bp. All the rfbE/fliC/SLT1/SLT2 probes specifically recognized Cy3-labeled fluorescent samples from O157:H7 strains, or strains containing O157 and H7 genes. No cross hybridization of O157:H7 fluorescent samples occurred in other probes. Non-O157:H7 pathogens failed to yield any signal under comparable conditions. If the Cy3-labeled fluorescent product of O157 single PCR was diluted 50-fold, no signal was found in agarose gel electrophoresis, but a positive signal was found in microarray hybridization. CONCLUSION: Microarray analysis of O157:H7 is a rapid, specific, and efficient method for identification and detection of bacterial pathogens.

Incidence of HBV variants with a mutation at nt551 among hepatitis B patients in Nanjing and its neighbourhood.

AIM: To investigate the epidemiology of hepatitis B virus (HBV) strains with a mutation at nt551 in surface gene among hepatitis B patients in Nanjing and its neighbourhood. METHODS: By using mutation-specific polymerase chain reaction (msPCR) established by our laboratory for amplifying HBV DNAs with a mutation at nt551, 117 serum samples taken from hepatitis B patients were detected. RESULTS: The results showed that 112 samples were positive for nt551A, 4 samples were positive for nt551G. One sample was positive for nt551T. No nt551C of HBV DNA was found. The incidence of HBsAg mutants with G, C, T, A at nt551 among 117 samples was 3.42%, 0%, 0.85%, 95.73%, respectively. CONCLUSION: In Nanjing and its neighbourhood, hepatitis B patients are mainly infected with wild genotype HBV. The incidence of mutants with a mutation at nt551 in HBV genome is significantly lower than that in wild genotype HBV DNA (P<0.01). The necessity of adding components of HBsAg mutants to HBV vaccine needs further investigation.

A mutation specific polymerase chain reaction for detecting hepatitis B virus genome mutations at nt551.

AIM: The hepatitis B surface antigen (HBsAg) is considered to be one of the best markers for the diagnosis of acute and chronic HBV infection. But in some patients, this antigen cannot be detected by routine serological assays despite the presence of virus. One of the most important explanations for the lack of detectable HBsAg is that mutations which occur within the "a" determinant of HBV S gene can alter expression of HBsAg and lead to changes of antigenicity and immunogenicity of HBsAg accordingly. As a result, these mutants cannot be detected by diagnosis assays. Thus, it is essential to find out specific and sensitive methods to test the new mutants and further investigate their distribution. This study is to establish a method to investigate the distribution of the HBsAg mutant at nt551. METHODS: A mutation specific polymerase chain reaction (msPCR) was established for amplifying HBV DNA with a mutation at nt551. Four sets of primer pairs, P551A-PPS, P551G-PPS, P551C-PPS and P551T-PPS, with the same sequences except for one base at 3' terminus were designed and synthesized according to the known HBV genome sequences and the popular HBV subtypes, adr and adw, in China. At the basis of regular PCR method, we explored the specific conditions for amplifying HBV DNAs with a mutation at nt551 by regulating annealing temperature and the concentration of these primers. 126 serum samples from patients of hepatitis B were collected, among which 16 were positive for HBV S DNA in the nested PCR amplification. These 16 HBV S DNAs were detected by using the msPCR method. RESULTS: When the annealing temperature was raised to 71 degrees, nt551A and nt551G were amplified specifically by P551A-PPS and P551G-PPS; At 72 degrees and 5 pmole of the primers (each) in reaction of 25 microl volume, nt551C and nt551T were amplified specifically by P551C-PPS and P551T-PPS. 16 of HBV S gene fragments were characterized by using this method. 14 of them were positive for nt551A, one was positive for nt551G, and the other one was positive for nt551T. The results were confirmed by nucleotide sequencing. CONCLUSION: The mutation specific polymerase chain reaction is a specific and sensitive method for detecting the mutations of HBV genome at nt551.

A novel hepatitis B virus mutant with A-to-G at nt551 in the surface antigen gene.

AIM: Hepatitis B surface antigen (HBsAg) mutant of hepatitis B virus (HBV) is one of the important factors that result in immune escape and cause failure of immunization. In this study we reported and characterized a novel HBV mutant with A-to-G at nt551 and intended to provide theoretical data for prevention of HBV infection in China. METHODS: A methodology comprising polymerase chain reaction (PCR) amplifying, M13 bacteriophage cloning and nucleotide sequencing was used to analyze the sera of the pediatric patient who was hepatitis B (HB) immune failure. Expression plasmids containing the mutant S gene and a wild-type (adr) S gene were constructed respectively and the recombinant HBsAg were expressed in COS-7 cells under the regulation of SV40 early promoter. The recombinant proteins were investigated for their immunological reactivity with different monoclonal antibodies (mAb) against "a" determinant and vaccine-raised human neutralizing antibodies. RESULTS: It was found that there was a new point mutation at nt551 of the HBV (adr) genome from A to G, leading to a substitution of methionine (Met) to valine (Val) at position 133 in the "a" determinant of HBsAg. Compared to the wild-type HBsAg, the binding activity of the mutant HBsAg to mAbs (A6, A11 and S17) and to vaccine-raised human anti-hepatitis B surface antibody (anti-HBs) decreased significantly. CONCLUSION: According to the facts that the patient has been immunized with HB vaccine and that the serum is anti-HBs positive and HBsAg negative, and based on the nucleotide sequence analysis of the mutant HBV S gene and its alteration of antigenicity, the HBV is considered to be a new vaccine-induced immune escape mutant different from the known ones.