Use of PCR Targeting of Internal Transcribed Spacer Regions and Single-Stranded Conformation Polymorphism Analysis of Sequence Variation in Different Regions of rRNA Genes in Fungi for Rapid Diagnosis of Mycotic Keratitis

Abstract

The increased incidence of fungal infections in the recent past has been attributed to the increase in the number of human immunodeficiency virus-positive and AIDS patients. Early diagnosis of mycoses in patients is crucial for prompt antifungal therapy. Immunological methods of diagnosis have not been found to be satisfactory, and recent research has been diverted to the use of PCR for the sensitive and early diagnosis at the molecular level. In the present study we targeted different regions of the rRNA gene to diagnose cases of mycotic keratitis and identify the causal agents. Six fungus-specific primers (primers ITS1, ITS2, ITS3, ITS4, invSR1R, and LR12R) were used, and the amplified products were analyzed by single-stranded conformation polymorphism (SSCP) analysis. Dendrograms of these SSCP patterns, prepared on the basis of Jaccard's coefficient, indicated that the PCR products obtained with primer pair ITS1 and ITS2 were the best for the identification of fungi. The results were confirmed by sequencing of the PCR products, and the approach was successfully tested experimentally for the detection of mycotic keratitis caused by Aspergillus fumigatus and was used for the diagnosis of fungal corneal ulcers in patients.Mycotic keratitis or fungal corneal infections have a worldwide distribution, and the incidence is higher in tropical and subtropical countries (8). More than 105 species of fungi belonging to 56 genera have been reported to cause oculomycosis. However, species of Fusarium, Aspergillus, Candida, and other hyaline and dematiaceous hyphomycetes are the usual isolates from patients with mycotic keratitis (20). The management of keratomycosis depends on rapid identification of the causal agent. The diagnosis is often delayed because of the poor availability of infected material from the cornea and the slow growth of a large number of fungi in routinely used culture media, and therefore, early intervention is not always possible and the patient's vision is often lost. In general, the diagnosis of fungal corneal ulcer is dependent on Gram and Giemsa staining, which have low sensitivities of about 50 to 80% (4). Recent advances in molecular biology techniques have opened the door for culture-independent diagnostic methods. Immunological detection (13) and identification by use of distinctive metabolites (2) and nucleic acid probes (7,25) are the tools most often used for diagnosis. One such technique is PCR, which has been shown to be useful for the culture-independent diagnosis of various microbial infections, including mycoses (10,11,22). To date, a few cases of mycotic keratitis have successfully been diagnosed by PCR (5, 6).Ribosomal DNA is the most conserved region in the genome, with capabilities of phylogenetic divergence (14). The whole rRNA gene contains a small subunit (SSU) 18S rRNA, 5.8S rRNA, and a large subunit (LSU) 28S rRNA. Internal transcribed spacer (ITS) region I (ITSI) and ITSII are more variable than the rest of the ribosomal gene subunits and are found between SSU rRNA and 5.8S ...

Evolutionary history of the seagrass genus Posidonia

“…The complete ITS region of the nuclear ribosomal DNA (ITS1, 5.8S rDNA and ITS2) was amplified using the primers ITS1 and ITS4 (White et al 1990) following the same conditions described in (Kumar & Shukla 2005). The 25 µl reaction mixture contained 100 µM dNTPs, 0.5 µM of each primer, 1× PCR buffer, 1.5 mM MgCl 2 , 5 µl of template DNA (with a final concentration of about 10 ng µl -1 ) and 1 U of Taq polymerase.…”

Морфолого-культуральні властивості рідкісного лікарського гриба Sparassis crispa (Sparassidaceae, Polyporales)

“…1) які зберігаються у Колекції культур шапинкових грибів (ІВК) (Kumar and Shukla, 2005). В дослідженні використовували 20 добовий вегетативний міцелій штамів S. crispa.…”

“…Підготовлені заморожені зразки ДНК передавали для подальших молекулярних досліджень до лабораторії компанії L.F. Lambert Spawn Co. (Коатсвіль, Пенсильванія, США). Полімеразну ланцюгову реакцію (ПЛР) здійснювали в ампліфікаторі T100 TM Thermal Cycler (Bio-Rad Laboratories, Inc., Геркулес, США), використовуючи стандартний протокол для ділянок внутрішнього транскрибуючого спейсера (ITS) у грибів (Kumar and Shukla, 2005). Для підтвердження успішності проведеної ампліфікації здійснювали гель-електрофорез ампліфікованих фрагментів ДНК в 1%-му агарозному гелі на основі буферу 1xSB (борат натрію, Faster Better Media LLC, Хант Велей, США) при напрузі 120V y PowerPac TM Basic (Bio-Rad Laboratories, Inc., Геркулес, США).…”

Perfil epidemiológico das ceratomicoses atendidas no HC-UNICAMP

“…Alguns autores recomendam a introdução de terapia antifúngica apenas após a confirmação laboratorial, com base na maior prevalência das infecções bacterianas (8) . Observamos na prática, porém, que há um alto número de exames falso-negativos, acarretando demora da introdução da terapia antifúngica adequada (9,10) . Devido a essa dificuldade no diagnóstico laboratorial, a história clínica e o exame físico ganham especial importância.…”