Evaluation of synthetic DNA probes for confirmation of Clostridium perfringens enterotoxin gene PCR products.

A new diagnostic reagent was developed that is capable of detecting the presence of Clostridium perfringens rapidly and accurately compared to the conventional methods. C. perfringens enterotoxin (cpe) gene is the gene of interest since it encodes the enterotoxin responsible for food poisoning. Two new cpe-specific labeled DNA probes were evaluated using Southern and dot blot hybridization. Bacterial DNA was amplified by a duplex PCR procedure. The results showed that 40 enterotoxin producing C. perfringens strains generated two bands of amplicons with sizes of 420 and 280 bp, whereas 40 non-enterotoxin producing strains produced a single band of 280 bp on agarose gel-electrophoresis. No bands were observed from 32 strains of Clostridium spp and other bacteria. Southern blot analysis using either cpe-specific DNA or oligonucleotide probe showed hybridization specifically to the 420 bp band in enterotoxin-positive C. perfringens. On the dot blot membrane, both cpe-specific DNA and oligonucleotide probes were able to hybridize specifically with the corresponding DNA templates but with different efficacy (100% vs 91.1%).

Development and comparison of the real-time amplification based methods--NASBA-Beacon, RT-PCR taqman and RT-PCR hybridization probe assays--for the qualitative detection of sars coronavirus.

The aim of this study was to develop a rapid, sensitive and robust procedure for the qualitative detection of SARS coronavirus RNA. Three unique detection formats were developed for real-time RNA amplification assays: a post amplification detection step with a virus-specific internal capture probe based on Taqman (RT-PCR TaqMan assay), hybridization probe (RT-PCR hybridization probe assay) and a real-time assay with virus-specific molecular beacon probes (NASBA-Beacon assay). The analytical sensitivity or reproducibility of the test results among those three assays was compared. All assays yielded results by detecting SARS coronavirus targeting the BNI-1 region in less than 2 hours. RNA detection by all the formats was unaffected by the presence of human sputum. The limits of detection were at least 10 copies of input RNA for both RT-PCR formats (RT-PCR TaqMan and RT-PCR hybridization probe assays), while the NASBA-Beacon assay could detect as little as 1 copy per reaction, with high reproducibility of the coefficient of variation (CV) of <10. These results demonstrate that real-time NASBA provides a rapid and sensitive alternative to RT-PCR for the routine qualitative assay of sputum for SARS corona viral RNA detection.

Nucleic acid probes in periodontal diagnosis.

Periodontal Disease is considered as an infection caused by a variety of microorganisms. Considering the vast range of microbial species involved in the causation of periodontal diseases, a specific diagnostic procedure, to identify the various organisms involved is a major necessity. A number of diagnostic procedures, including microbiological and immunological have been utilized in the diagnosis of periodontal disease. The DNA probe technology provides both a sensitive and specific assay and also alleviates concern for transport of the fastidious microorganisms in clinical samples, fulfills the need for a specific and rapid test, that does not require the preservation of viable microorganisms. DNA probes are now being used to identify the various putative pathogen's including; A. actinomycetemcomitans, P. gingivalis, P. intermedia, B. forsythus, etc. Besides DNA probes have also been proven an advantage over the various other available procedures in periodontal diagnosis.

Use of PCR/DNA probes to identify circumsporozoite genotype of Plasmodium vivax in China.

The paper reports the result of identifying cirumsporozoite (CS) genotype of Plasmodium vivax by using PCR/DNA probe labeled with biotin. The sensitivity of this method to detect patient blood samples was 0.2 parasite/microl and also with high specific to P. vivax. CS genes from 52 blood samples collected from patients with P. vivax in Hainan and Yunnan Provinces were amplified by PCR and 49 were positive by gel-e electrophoresis analysis, positive rate was 94%. Then the amplified CS genes further were probed with special oligoprobes (PV210 and PV247) that hybridized with the predominant CS repeat region and the variant CS repeat region. The results showed 46 (88.5%) PV210 positive and 6 (11.5%) PV247 positive; 2 hybridized with both probes. The variant genotype was present only in samples from Yunnan Province. The above results showed that the PCR/DNA probe labeled with biotin was highly sensitive and specific to P. vivax and found a CS variant genotype of P. vivax in Yunnan Province of China.

Dengue vector mosquitos at a tourist attraction, Ko Samui, in 1995.

On Ko Samui, Thailand there were two epidemics of dengue hemorrhagic fever (DHF) in 1966 and 1967, followed by endemics up to 1994. Aedes aegypti and Aedes albopictus were the vectors. From January to July 1995, 51 cases of DHF were reported, out of these were many foreigners who still suffer from dengue fever and return home with negative impression. We carried out an entomological survey around the island and collected the mosquitos to detect dengue virus by digoxigenin-cDNA probe. The data revealed that Aedes aegypti and Aedes albopictus still were abundant and some were infected with dengue virus. Visual larval survey indices (HI, CI and BI) were 90.4, 61.3 and 301.3 respectively. Biting rate (BR) of Aedes mosquitos was high, the average indoor and outdoor BR were 9.7 and 100.8 mosquitos/man-hour. From 13 pools of mosquitos, 8 strains of dengue virus were detected (61.5%). The results may encourage the local authorities to improve vector surveillance and control before the famous island becomes an unpleasant island.

Hemolysins and plasmid profiles of Vibrio parahaemolyticus.

Forty clinical isolates of Vibrio parahaemolyticus were studied for the production of the thermostable direct hemolysin (TDH), and the TDH-related hemolysin (TRH) including the respective encoding genes, tdh and trh. The presence of TDH and its encoding genes were found amongst 95% of the strains, whereas the TRH was absent amongst these isolates. Thirty-two isolates were found to be plasmid-free, whereas eight isolates possessed plasmids with sizes ranging from 2.4 > or = 23 kb. Using a DNA probe coding for the homologous region of the tdh and trh, it was found that the tdh genes were present on the chromosomal DNA.

Shortcomings of some currently available DNA probes for malaria detection.

A non-radioactive DNA probe based-method for detecting malaria will greatly aid epidemiological studies. Using putative Plasmodium falciparum and P. vivax-specific 18S ribosomal RNA directed oligonucleotides, different enzymatic and chemiluminescent detection methods were attempted without success. The sensitivity of the corresponding 32P-labelled probes was found to be inadequate. A published procedure based on chemiluminescent detection of repetitive DNA sequences of P. falciparum was found to be adequately sensitive but lacking in specificity.

Recent advances in the application of molecular biology in filariasis.

Monitoring of filarial parasites in the host and vector has traditionally depended on morphological identification. Recently, species-specific DNA probes have been developed for Brugia malayi, Brugia pahangi and Wuchereria bancrofti. Repeated DNA sequences are useful in developing DNA probes because they evolve more rapidly then coding sequences and their high copy number increases the sensitivity of detection. The Hhal repeated DNA family represents 12% of the total B. malayi DNA. This DNA family is present in species of Brugia (B. malayi, B. timori and B. pahangi) but not W. bancrofti. Sequence analysis of the repeated DNA in B. malayi and B. pahangi has allowed construction of two species-specific DNA probes. These probes were used in a double blind field study in Indonesia. Microfilariae (mf) from infected cats and humans were identified by classical morphological methods and DNA probes. Agreement was found in 98.6% of the 642 samples tested by the two different techniques. Besides mf identification DNA probes can be used to determine the species of infective larvae (L3s) in infected mosquitos. This is useful because the L3s have similar morphology. DNA probes for the identification of W. bancrofti have recently been developed and are in the initial stages of testing in China (Piessens, personal communication) and Egypt (Williams, personal communication). An alternative approach for identification of infected individuals is to detect specific parasite antigens in circulation. A WHO initiative to use either an antigen or antibody assay to replace night blood is presently underway. This approach, if successful would not require the presence of microfilariae, but could detect occult infections.

Diagnosis of tuberculosis by polymerase chain reaction.

For the diagnosis of extrapulmonary tuberculosis in adults and all forms of tubercular infections in children, microscopic and cultural techniques have been shown to be inadequate. Many serological techniques have been employed for non culture diagnosis of tuberculosis. Early promising results have repeatedly given way to subsequent findings of non-specificity. Major mycobacterial antigens have been shown to be heat shock proteins which are highly conserved in nature. DNA probes for tuberculosis are specific but have a sensitivity equivalent to AFB smear examination. Polymerase Chain Reaction (PCR) with its ability to selectively amplify DNA fragments of interest offers a potentially powerful technique for the rapid, specific and sensitive diagnosis of tuberculosis. Samples from partially treated patients could be culture negative but can be detected by PCR.

New approaches to the diagnosis of tuberculosis in childhood with special reference to neurotuberculosis.

Bacteriological diagnosis of tuberculosis in childhood is often unsuccessful owing to the difficulty in obtaining suitable specimens. Many attempts have been made to diagnose tuberculosis immunologically but with very limited success. Positive tuberculin reactions may be the result of nonspecific sensitization while negative reactions occur in undernourished children. Serodiagnostic tests suffer from problems of specificity, even when very specific antigens are used, and are often least helpful in diagnostically difficult cases. Detection of antigen has proved to be of more value, especially with clean specimens such as cerebrospinal and pleural fluids. Detection of specific components of Mycobacterium tuberculosis by linked gas chromatography and mass spectroscopy is very sensitive and specific but the equipment is very costly. Detection of specific DNA sequences of M. tuberculosis in specimens by use of labelled 'DNA probes' is rather insensitive although the sensitivity may be increased greatly by use of the polymerase chain reaction to amplify small amounts of the specific DNA. Non specific indicators of tuberculosis are generally unhelpful although the bromide partition test and assay of the enzyme adenosine deaminase in cerebrospinal fluid appear to be of value in the diagnosis of tuberculous meningitis. More research is required to develop a simple, specific and automated test for tuberculosis in childhood.