Vibrio cholerae has caused severe outbreaks of cholera worldwide with thousands of recorded deaths annually. Molecular diagnosis for cholera has become increasingly important for rapid detection of cholera as the conventional methods are time-consuming and labour intensive. However, traditional PCR tests still require cold-chain transportation and storage as well as trained personnel to perform, which makes them user-unfriendly. The aim of this study was to develop a thermostabilized triplex PCR test for cholera which is in a ready-to-use form and requires no cold chain. The PCR test specifically detects both toxigenic and non-toxigenic strains of V. cholerae based on the cholera toxin A (ctxA) and outer-membrane lipoprotein (lolB) genes. The thermostabilized triplex PCR also incorporates an internal amplification control that helps to check for PCR inhibitors in samples. PCR reagents and the specific primers were lyophilized into a pellet form in the presence of trehalose, which acts as an enzyme stabilizer. The triplex PCR was validated with 174 bacteria-spiked stool specimens and was found to be 100 % sensitive and specific. The stability of the thermostabilized PCR was evaluated using the Q 10 method and it was found to be stable for approximately 7 months at 24 6C. The limit of detection of the thermostabilized triplex PCR assay was 2¾10 4 c.f.u. at the bacterial cell level and 100 pg DNA at the genomic DNA level, comparable to conventional PCR methods. In conclusion, a rapid thermostabilized triplex PCR assay was developed for detecting toxigenic and non-toxigenic V. cholerae which requires minimal pipetting steps and is cold chain-free.
The lymphatic dwelling filariae Wuchereria bancrofti and Brugia malayi are estimated to infect approximately 119 million people worldwide (8). Affected individuals may suffer from clinical manifestations such as elephantiasis, hydrocoele, and adenolymphangitis or may have clinically silent manifestations of infection that are associated with subtle abnormalities of the lymphatic system. A diagnostic technique based on direct demonstration of the parasite or parasite products such as circulating filarial antigen (CFA) is the only definite way to detect infection in humans.Various groups of researchers have developed diagnostic tests that detect and quantify CFA (1, 3 5, 10, 13, 15, 18)
SummaryOBJECTIVE To quantify circulating antigens in individuals with lymphatic filariasis by means of an ELISA using blood on filter strips. METHOD Circulating antigens in filarial patients and normal individuals living in an area endemic for W. bancrofti infection in Madras, India were estimated using a monoclonal antibody-based ELISA. RESULTS All microfilaraemics showed positivity to circulating antigens whereas people with chronic pathology and 80% of the endemic normals tested negative. The antigen levels in the blood collected in the night and during day time showed positivity and there was no difference in the antigen concentration. The results of the antigen levels collected onto filter strips correlated with their corresponding plasma antigen levels (r ϭ 0.83). In microfilaraemics, DEC treatment did not alter the levels of circulating antigens for up to one month. CONCLUSIONS We conclude that this monoclonal antibody-based ELISA using filter strips may be used in day time and replace the existing routine night blood surveys in our endemic area in India.
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