In the search for universal vaccine candidates for the prevention of avian influenza, the non-structural (NS)-1 protein of avian influenza virus (AIV) H5N1 has shown promising potential for its ability to effectively stimulate the host immunity. This study was aimed to produce a bacterial expression plasmid using pRSET B vector to harbour the NS1 gene of AIV H5N1 (A/Chicken/Malaysia/5858/2004 (H5N1)) for protein expression in Escherichia coli (E. coli). The NS1 gene (687 bp) was initially amplified by polymerase chain reaction (PCR) and then cloned into a pGEM-T Easy TA vector. The NS1 gene was released from pGEM-T-NS1 using EcoRI and XhoI restriction enzymes (RE). The pRSET B vector was also linearized using the same RE. The digested NS1 gene and linearized pRSET B were ligated using T4 DNA ligase to form the expression plasmid, pRSET B-NS1. The NS1 gene sequence in pRSET B-NS1 was confirmed by DNA sequencing. To prepare recombinant bacterial cells for protein expression in the future, pRSET B-NS1 was transformed into E. coli strain BL21 (DE3) by heat-shock. Colonies bearing the recombinant plasmid were screened using PCR. The DNA sequencing analysis revealed that the NS1 gene sequence was 97% homologous to that of AIV H5N1 A/Chicken/Malaysia/5858/2004 (H5N1). These results indicated that the NS1 gene of influenza A/Chicken/Malaysia/5858/2004 (H5N1) was successfully amplified and cloned into a pRSET B vector. Bacterial colonies carrying pRSET B-NS1 can be used for the synthesis of NS1-based influenza vaccine in the future and thereby aid in the prevention of avian influenza.
Infeksi kulat sistemik yang paling kerap berlaku pada pesakit hospital adalah infeksi kandidiasis yang disebabkan oleh Candida spp. Pendiagnosan infeksi ini melalui pengkulturan dan ujian serologi mengambil masa ataupun kurang sensitif dan spesifik. Oleh itu, tindak balas rantai polimerase (PCR) yang mengesan DNA kulat telah diperkembangkan untuk mendapatkan diagnosis yang cepat dan tepat. Dalam kajian ini, keupayaan asai PCR ‘seminested’ untuk mengesan infeksi sistemik Candida albicans pada haiwan makmal telah ditentukan. Tikus dewasa Sprague-Dawley diinfeksi dengan C. albicans secara suntikan intravena sel yis tersebut. Darah tikus diperolehi setiap 3 atau 4 hari untuk ekstraksi DNA. Setiap minggu, 3 ekor tikus dikorbankan dan organ-organ dalamannya dikultur untuk memastikan kehadiran infeksi sistemik C. albicans. Tempoh kajian ini adalah selama 4 minggu (28 hari). PCR ‘seminested’ dijalankan ke atas sampel DNA dengan menggunakan primer fungus universal, iaitu ITS1 dan ITS3 serta primer spesifik C. albicans (CALB1). Produk PCR yang terhasil dikesan dengan elektroforesis gel agaros. Asai PCR ‘seminested’ berjaya mengesan DNA C. albicans di dalam sampel darah haiwan terinfeksi dari hari ke-2 sehingga hari ke-25 pos-infeksi secara tekal. Pengkulturan organ ginjal, hati dan limpa menunjukkan haiwan tersebut diinfeksi secara sistemik sehingga hari ke-21 pos-infeksi dan telah pulih (keputusan kultur negatif) pada hari ke-28 pos-infeksi. Kesimpulannya, kajian ini menunjukkan PCR ‘seminested’ adalah satu kaedah yang berupaya mengesan infeksi sistemik Candida albicans sepanjang tempoh infeksi. Oleh itu, asai PCR ini mempunyai nilai sebagai kaedah diagnosis yang berkesan dan juga mampu menentukan prognosis apabila memantau status infeksi.
Ergosterol, a component of fungal cell membrane, has been frequently detected as an indicator of fungal presence and mass
in environmental samples like soil. However, its detection in major pathogenic fungal species has not been investigated.
In this study, the ergosterol contents of ten pathogenic fungal species were determined. Liquid chromatography was used
for the detection and quantification of ergosterol extracted from fungal broth cultures. Results showed that ergosterol
eluted as a single, well resolved peak in the chromatogram profiles of all tested fungi. Based upon relative amounts of
ergosterol produced per fungal mycelial dry weight, three groups of fungal pathogens were identified, namely low ergosterol
(Aspergillus niger, Candida albicans and Cryptococcus neoformans at 4.62, 6.29 and 7.08 µg/mg, respectively), medium
ergosterol (Fusarium solani, Aspergillus fumigatus, Mucor sp., Penicillium sp., Cryptococcus gattii and Rhizopus sp.
at 9.40, 10.79, 10.82, 11.38, 12.60 and 13.40 µg/mg, respectively), and high ergosterol (Candida tropicalis at 22.84 µg/
mg), producers. Ergosterol was not detectable in bacterial samples, which were included as controls. This first report on
ergosterol detection in major pathogenic fungal species indicates that ergosterol may be used as a biomarker to diagnose
invasive fungal infections in clinical samples.
Invasive fungal infections (IFIs) have risen dramatically in recent years among high risk immunocompromised patients.
Rapid detection of fungal pathogens is crucial to timely and accurate antifungal therapy. Two multiplex polymerase
chain reaction (PCR) assays were developed to detect major fungal species that cause invasive infections and identify
resistant species. Genus specific primers for Candida, Aspergillus, Fusarium and species specific primers for Candida
glabrata, Candida krusei and Aspergillus terreus which are known to be clinically resistant species, were designed from
the internal transcribed spacer (ITS) regions of ribosomal ribonucleic acid (rRNA) gene complex. Both assays were
performed simultaneously to promote rapid detection of fungal isolates based on distinct amplicon sizes. Inclusion of the
universal fungal primers ITS 1 and ITS 4 in the genus specific assay produced a second amplicon for each isolate which
served to confirm the detection of a fungal target. The limit of detection for the genus specific assay was 1 nanogram
(ng) deoxyribonucleic acid (DNA) for Aspergillus fumigatus and Candida albicans, 0.1 ng DNA for Fusarium solani, while
the species-specific assay detected 0.1 ng DNA of A. terreus and 10 picogram (pg) DNA of C. krusei and C. glabrata. The
multiplex PCR assays, apart from universal detection of any fungal target, are able to detect clinically important fungi
and differentiate resistant species rapidly and accurately, which can contribute to timely implementation of effective
antifungal regime.