Establishment and application of a PCR-ELISA assay for the detection of seasonal influenza A virus subtypes H1 and H3 and influenza B virus / 中华微生物学和免疫学杂志

Objective To develop a PCR-ELISA assay for the rapid, specific and sensitive detec-tion of human seasonal influenza virus ( H1, H3 and B) by using molecular biological and immunological methods in combination.Methods The primers were designed according to the genes encoding the matrix protein ( M) , the H1 and H3 hemagglutinin ( HA) of influenza A virus and the nonstructural proteins ( B-NS) of influenza B virus and then were labeled with biotin.The PCR products were detected by ELISA by use of an internal catching probe labeled with DIG.Results The minimum copy numbers of genes encoding the M, H1, H3 and B-NS proteins detected by the established assay were 1.43?103 , 8.67?102 , 3.86?103 and 5.45?103 copies/μl, respectively, which indicated that the PCR-ELISA assay was about 10 times more sensitive than agarose gel electrophoresis in the detection of PCR products.No cross-reactions between the different subtypes of influenza virus or different species of virus were observed.Moreover, a total of 104 clin-ical specimens of influenza virus were examined by the PCR-ELISA assay, the results of which were consist-ent with those of the virus isolation method.Conclusion The newly developed PCR-ELISA assay was a highly sensitive and specific method for the rapid detection and subtyping of influenza virus, suggesting the possibility of using it in laboratory for the surveillance and detection of influenza virus.

Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA

Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS) Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins 1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.

Comparison of Rifampicin Resistance in Mycobacterium tuberculosis Isolates by Multiplex Allele Specific PCR (MAS-PCR) with Enzyme Linked Immunosorbent Assay (PCR-ELISA).

Aims: Detection of drug resistance M. tuberculosis isolates is one of the most important strategies to control the disease. Nowadays, with advances in molecular technology, various methods are available to detect drug resistant M. tuberculosis strains such as those based on capture specific probes. In this study, we aimed to investigate the frequency of mutation in the M. tuberculosis -rpoB gene by Polymerase Chain Reaction based on Enzyme Linked Immuno Sorbent Assay (PCR-ELISA) detection. Methodology: Thirty-three culture positive isolates were randomly selected for this study. All the isolates were subjected to a drug Susceptibility Test (DST) using the proportion method. Then the ability and the efficiency of Multiplex Allele Specific PCR (MAS-PCR) and PCR-ELISA to detect Rif resistant (Rifr) M. tuberculosis isolates was compared and evaluated. Results: Mutation of rpoB gene was detected in 19/33 isolates (57.6%) by PCR-ELISA. Hybridization with the specific mutant probes 516 and 526 codon occurred in 1/33 isolates each (3% respectively). Hybridization with the specific mutant probe 531 occurred in 13/33 isolates (39.4%). Three isolates (9.2%) showed simultaneous mutation in codons 516 and 531. The sensitivity and specificity of MAS-PCR in comparison to the Proportional Method was 100%. On the other hand, PCR-ELISA showed 75% sensitivity and 69.2% specificity. The positive predictive value for the PCR-ELISA method was 78.9% and the negative predictive value was 64.3%.The general efficacy of test was 72.7%. Conclusion: The study showed that the sensitivity and specificity of PCR-ELISA to detect mutations in the rpoB gene in Drug Resistant strains was low. Furthermore, this proved to be a complex, time consuming and expensive test. Therefore, this test is not recommended for determining Rifampicin resistance in M. tuberculosis strains.

Methodological study of quantitative detection of Mycobacterium tuberculosis by immunomagnetic capture combined with PCR-ELISA / 国际检验医学杂志

Objective To establish a quantitative detection method for Mycobacterium tuberculosis by immunomagnetic capture combined with PCR-ELISA detection system with double internal standards(IMC-PCR-ELISA) .Methods The immunomagnetic (Dynabeads? ) which could specifically capture Mycobacterium tuberculosis were prepared .According to Mtp40 and IS6110 gene sequence of Mycobacterium Tuberculosis ,2 pairs of primers(upstream primer was modified with Biotin at 5′end) ,2 same-length mutant fragments with PCR amplified fragments ,and 3 capture probes(modified with digoxigenin at 3′end) were designed .Myco-bacterium tuberculosis were captured by immunomagnetic ,then detected by PCR-ELISA with double internal standards .Results The IMC-PCR-ELISA method could yield quantitative results in about 4 h with a detection limit at 5 × 103 copies/mL .There was a fine linear relationship between the copies of Mtp40(IS6110)in fact and in the calculation through formula when the concentrations of low internal standards were 30-70 copies/mL and the concentrations of high internal standards were 8 000-12 000 copies/mL (r2 =0 .998) .No nonspecific amplification was observed .Conclusion A rapid and quantitative method for the detection of Myco-bacterium tuberculosis was established successfully .The IMC-PCR-ELISA method was rapid ,sensitive ,secific and quantitative .

Evaluation of PCR-ELISA as a tool for monitoring transmission of Wuchereria bancrofti in District of Gampaha, Sri Lanka

Objective: To compare Wuchereria bancrofti (W. bancrofti) infection rates of Culexquinquefasciatus, using dissection and PCR-ELISA in two consecutive time periods (from 2007 to 2008 and from 2008 to 2009). Methods: Mosquitoes were collected in 30 sentinel and 15 non-sentinel sites in 15 Medical Officer of Health areas of Gampaha District known for the presence ofW. bancrofti transmission in two consecutive time period of 2007 to 2008 and 2008 to 2009. Captured mosquitoes were dissected to determine the W. bancrofti larvae (L1, L2, L3). PCR was carried out using DNA extracted from mosquito pools (15 body parts/pool) utilizing the primers specific for Wb-SspI repeat. PCR products were analyzed by hybridization ELISA using fluorescein-labeled wild type specific probes. The prevalence of infected/infective mosquitoes in PCR pools (3 pools/site) was estimated using the PoolScreenTM algorithm and a novel probability-based method.Results:Of 45 batches of mosquitoes dissected, W. bancrofti infected mosquitoes were found in 19 and 13 batches, with an infection rate of 13.29% and 3.10% with mean larval density of 8.7 and 1.0 larvae per mosquito for two study periods in the Gampaha District. Total of 405 pools of head, thorax and abdomen were processed by PCR-ELISA for each year. Of these, 51 and 31 pools were positive for W. bancrofti in the two study periods respectively. The association of dissection based prevalence rates with PCR based rates as determined by the Pearson correlation coefficient were 0.176 and 0.890 respectively for the two periods. Conclusions: Data indicate that PCR-ELISA is more sensitive than the traditional dissection techniques for monitoring transmission intensity.

Evaluation of PCR-ELISA as a tool for monitoring transmission of Wuchereria bancrofti in District of Gampaha, Sri Lanka

<p><b>OBJECTIVE</b>To compare Wuchereria bancrofti (W. bancrofti) infection rates of Culex quinquefasciatus, using dissection and PCR-ELISA in two consecutive time periods (from 2007 to 2008 and from 2008 to 2009).</p><p><b>METHODS</b>Mosquitoes were collected in 30 sentinel and 15 non-sentinel sites in 15 Medical Officer of Health areas of Gampaha District known for the presence of W. bancrofti transmission in two consecutive time period of 2007 to 2008 and 2008 to 2009. Captured mosquitoes were dissected to determine the W. bancrofti larvae (L1, L2, L3). PCR was carried out using DNA extracted from mosquito pools (15 body parts/pool) utilizing the primers specific for Wb-SspI repeat. PCR products were analyzed by hybridization ELISA using fluorescein-labeled wild type specific probes. The prevalence of infected/infective mosquitoes in PCR pools (3 pools/site) was estimated using the PoolScreen™ algorithm and a novel probability-based method.</p><p><b>RESULTS</b>Of 45 batches of mosquitoes dissected, W. bancrofti infected mosquitoes were found in 19 and 13 batches, with an infection rate of 13.29% and 3.10% with mean larval density of 8.7 and 1.0 larvae per mosquito for two study periods in the Gampaha District. Total of 405 pools of head, thorax and abdomen were processed by PCR-ELISA for each year. Of these, 51 and 31 pools were positive for W. bancrofti in the two study periods respectively. The association of dissection based prevalence rates with PCR based rates as determined by the Pearson correlation coefficient were 0.176 and 0.890 respectively for the two periods.</p><p><b>CONCLUSIONS</b>Data indicate that PCR-ELISA is more sensitive than the traditional dissection techniques for monitoring transmission intensity.</p>

Application of a Diagnostic Method Using Reverse Transcription-PCR ELISA for the Diagnosis of Enteroviral Infections / 대한진단검사의학회지

BACKGROUND: Enteroviruses are known as major pathogen for aseptic meningitis. Although rapid diagnosis for enteroviruses is very essential to exclude bacterial infections in patients with meningitis, classical diagnostic method based on virus isolation is not practicable for timely treatment of patients due to its laborious and time-consuming procedure. Recently molecular methodologies as alternatives are routinely used for rapid and sensitive diagnosis for enteroviruses infections. METHODS: Reverse transcription (RT)-PCR ELISA kit for targeting 5'non-coding region (NCR) with highly conserved genetic identity among all genotypes of enteroviruses was introduced in this investigation. RT-PCR ELISA was evaluated about sensitivity and specificity through virus isolation using clinical specimens from patients suspected of enteroviral infections and enteroviral isolates comparing with conventional RT-PCR identifying them. RESULTS: The detection limit of the RT-PCR ELISA was up to 10-100 folds higher than virus isolation using cell culture and conventional RT-PCR. On comparison between above two methods, the detection rate of RT-PCR ELISA for clinical specimens from patients with aseptic meningitis was 7% higher than that of conventional RT-PCR targeting 5'NCR (P=0.016). CONCLUSIONS: Our results suggest that RT-PCR ELISA developed in this study could be an alternative diagnostic method for the detection of enteroviral genome with high sensitivity and specificity.

Two-Step MS-PCR Combined With ELISA Method for the Detection of Drug Resistance Mutations in HIV-1 RT Gene / 中国生物工程杂志

Highly Active Anti-Retroviral Therapy (HAART) has effectively inhibited the prevalence of HIV-1 and reduced the death rate caused by AIDS. In recent years,the emergence of resistance-conferring RT gene mutations in HIV-1 strains has become the major reason for HAART failure. The detection of drug resistance is important for the HAART regimen choice and novel drug development. A novel assay for the detection of HIV-1 RT drug resistance mutations was developed. HIV-1 drug resistance and wild strains in B subtypes were investigated using Two-Step Mutagenically-Separated PCR (MS-PCR),and point mutations including M41L,K70R,K103N,Y181C,T215F were detected. A longer mutant type primer was designed,using microplates hybridization and ELISA technique to detect several point mutations within a mixed mutant-wild type population. The results indicate that the Two-Step MS-PCR is as sensitive and specific as that in the traditional MS-PCR and MS-PCR combined with ELISA can give a good P/N quotient with better sensitivity,low cost,relatively less time consumption and high-throughput screening. It will be used in clinic usage for the detection of HIV-1 drug resistance mutations as well as other point mutations.

Development of PCR-ELISA for Detection of HIV-1 in Biomedical Products / 대한진단검사의학회지

BACKGROUND: Biomedical products such as viral vaccines can be contaminated with hazardous viruses during manufacturing processes and storage, thus causing harmful side effects. To assure the safety of biomedical products, highly effective and sensitive methods should be available to detect contaminating viruses. In this study, we performed recovery tests to determine the limit of detection of HIV-1. METHODS: An HIV-1 plasmid preparation was serially diluted and spiked into various culture media (DMEM, RPMI-1640, IMDM, GICM, and SDM) containing 10% fetal bovine serum (FBS). The HIV-1 plasmid was detected by PCR alone or a combination of PCR and ELISA (PCR/ELISA). RESULTS: When spiked into DMEM, RPMI, and IMDM, less than 4x10(-2) ng of HIV-1 plasmid was not detectable as HIV-1 PCR products in agarose gel. Intra- and inter-assays (n=6) showed that the PCR-ELISA system could detect PCR products diluted as much as 1, 875 times from HIV-1 plasmid serially spiked in various media. CONCLUSIONS: The PCR/ELISA system can be useful for the detection of trace amounts of hazardous viruses which may be present as contaminants in biological products.

Development of Efficient Screening Methods for Hazardous HBV Virus in Vaccine Preparation

As viral vaccines sometimes induce the side effects which intimidate humans, it is urgently required to study about the side effects of viral vaccines. Most vaccines are derived from the biological forms. Hence it should be established the efficient and sensitive evaluation methods for the possibility of dangerous contaminants such as viruses and the stability of vaccines. A lots of vaccine products have been imported, or in sale or being developed in nation. Some of products may be mixed with the hazardous viruses derived from animal or human resources. Such hazardous viruses should be identified specifically and efficiently. Biological products are not permitted to distribute or obtain without presenting the result of absence experiment of hazardous viruses and prion and the validation data of inactivation experiment during processes. In order to detect viruses, there were TEM for viral particles, infectivity assays and detection methods for nucleic acids. However, these methods have defects such as insensitivity and inaccuracy. It should be considered to detect only the hazardous viruses with specificity, precision and accuracy during the processes of preparation, transportation and storage. It is noted to increase the detection limit in order to detect the minute hazardous factors during preparation processes for viral vaccines. This study is focused on the establishment of various sensitive PCR/ELISA methods for HBV virus which enhance the detection limit in order to detect the minute hazardous factors during preparation processes for viral vaccines.

Determination of telomerase activity in exfoliated cells in urine for the diagnosis of bladder carcinoma / 中华检验医学杂志

Objective In order to find a more sensitive and specific noninvasive assay for the diagnosis of bladder carcinoma, the authors tested the exfoliated cells from patient′s voided urine for the presence of telomerase activity and evaluated its clinical significance.Methods PCR-ELISA assay was used to determine the presence of telomerase activity in voided urine samples from patients with bladder carcinoma, fhematuria of benign causes and from normal, healthy volunteers.Results Telomerase activity was detected in 14 of 18 tumor samples, while none of the hematuria samples and normal, healthy volunteers′ samples. Conclusions Telomerase activity could be detected in most urine samples of bladder carcinoma patients and it is more sensitive and specific than other assays, therefore, would be a good diagnostic marker.

Telomerase PCR-ELISA Assay in Diagnosis of Bladder Tumor / 대한비뇨기과학회지

PURPOSE: The detection of telomerase activity is a new and useful method in diagnosis of bladder transitional cell carcinoma(TCC) in urine samples. But the detection method of telomerase activity is not easily performed in clinical settings because it uses radio-isotope and electrophoresis. We evaluated the test results of telomerase PCR-ELISA and compared them with the results of urinary cytology. MATERIALS AND METHODS: In order to evaluate the feasibility of telomerase PCR-ELISA method in bladder TCC, 36 bladder washing samples of patients with bladder TCC and 10 bladder washing samples of benign urologic diseases were examined for telomerase activity. RESULTS: The overall sensitivity and specificity of the telomerase test was 76.5%(26/36) and 80.0%(4/5). The sensitivity of telomerase test was higher than that of urinary cytology in low grade bladder TCC. Sensitivity of the telomerase test according to the nuclear grade of bladder TCC was 61.5% in grade I, 92.3% in grade II, 75% in grade III. In contrast, the sensitivity was 38.5% in grade I, 66.7% in grade II, 87.5% in grade III in urinary cytology. There was no correlation between the tumor stages and the sensitivity of telomerase test. CONCLUSIONS: We have shown that the sensitivity and specificity of telomerase PCR-ELISA method is similar to the results of telomerase tests previously reported using radioisotope. Furthermore, the telomerase test is more sensitive in detecting bladder tumor of low grade than urinary cytology. These findings suggest that telomerase PCR-ELISA method can be used conveniently and widely for the detection of bladder tumor in clinical practice.

Early Detection of Human Cytomegalovirus DNA by PCR-ELISA / 대한임상병리학회지

BACKGROUND: Human cytomegalovirus (CMV) infections are common and occasionally severe in newborns, immunocompromised hosts, cancer patients, and recipients of organ transplant. Consequently, sensitive and rapid methods for CMV detection are of great diagnostic value since antiviral drugs have become available, which might be more effective upon early administration. We evaluated a polymerase chain reaction and enzyme-linked immunosorbent assay (PCR- ELISA) to detect human CMV infection as an aid in making a prompt diagnosis and a determination of therapeutic efficacy. METHODS: CMV DNA was amplified by single PCR, using primers chosen from genomic regions (major immediate-early [MIE] protein coding region), and the microwell plate hybridization assay was performed for specific detection of 5'-biotinylated PCR products using CMV-specific probes labeled with digoxigenin. A total of 35 clinical specimens from 14 patients who were suspected CMV infectious state was analyzed by PCR-ELISA and its results were compared with those of serum anti-CMV IgM, shell vial culture assay and PCR. RESULTS: The sensitivity for detection of PCR-amplified CMV DNA by the ELISA was 102 copies, which was ten-fold greater than ethidium bromide staining of agarose gels. The positive rates of 35 clinical specimens by serology, shell vial culture assay, PCR and PCR-ELISA were 37.9%, 40.0%, 60.0% and 68.6%, respectively. The OD ranges of 24 positive specimens by PCR-ELISA were from 0.042 to above 2.5. In follow-up studies of two patients with bone marrow transplantation, positive CMV results by PCR-ELISA earlier than by other methods including serologic method, shell vial culture assay and PCR. CONCLUSIONS: These results reveal that PCR-ELISA may show higher sensitivity and positive rate than serologic method, shell vial culture assay and conventional PCR. PCR-ELISA can be useful to manage CMV infection rapidly in patients at risk.

Detection of Pneumocystis carinii DNA in Rats by PCR-ELISA / 中国寄生虫学与寄生虫病杂志

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Detection of main flaviviruses using polymerase chain reaction and microwell plate hybridization / 解放军医学杂志

Objective To establish a specific,sensitive and applied method for the detection and differentiation of dengue virus types Ⅰ-Ⅳ,Japanese encephalitis virus and yellow fever virus.Methods Based on the genomes sequence analysis,6 pairs of primers were designed.The special capture probes of dengue virus types Ⅰ-Ⅳ,Japanese encephalitis virus and yellow fever virus were amplified,cloned and sequenced.Then the microwell plates were precoated using these capture probes,and the forward primers were labeled using biotin.The samples were then amplified using the biotin labeled forward primers and reward primers.The microwell plate hybridization was processed for detecting and differentiating the virus.The precoated DNA concentration,precoated time,hybridization temperature and hybridization time were optimized carefully.Results The A value of positive samples were over 0.5,while the average A value of the negative samples was less than 0.1.The S/N value exceeded 10.0.Sensitivity experiment suggested the method of PCR-ELISA could detect the virus RNA in 107 times dilution,while RT-PCR could detect the virus RNA in only 106 times dilution.The stability experiment of PCR-ELISA using DVⅠ suggested that the within-batch coefficient of variation was 6.21%,the between-batch coefficient of variation was 9.92%;the within-batch coefficient of variation in negative control was 1.92%,and the between-batch coefficient of variation in negative control was 3.68%.No visible changes were found on the performance of the coated microwell plates when stored in 4℃for 6 monthes.Conclusion PCR-ELISA is a more sensitive and specific method than RT-PCR is in the early detection and type identification of dengue Ⅰ-Ⅳ types virus,Japanese encephalitis virus and yellow fever virus.