Development and evaluation of loop-mediated isothermal amplification (LAMP) for the rapid diagnosis of Penicillium marneffei in archived tissue samples.
Penicillium marneffei is the etiologic agent of a severe systemic disease in immunocompromised hosts in Southeast Asia. In the present study, a novel method, known as loop-mediated isothermal amplification (LAMP), is described for the rapid and specific detection of the species, using a primer set derived from the internal transcribed spacer (ITS) region of the rRNA gene. Amplification products can be detected macroscopically by visual inspection in vials using SYBR Green I as well as by electrophoresis on agarose gel. The LAMP assay resulted in specific amplification of P. marneffei ITS using pure cultures after a 1-h reaction at 65 degrees C in a water bath; no cross-reactivity with other fungi including other biverticillate penicillia was observed. The detectable DNA limit was two copies. In addition, specific amplification was achieved using paraffin wax-embedded tissue samples from patients with penicilliosis marneffei and tissue samples from bamboo rats. The method provides a powerful tool for rapid diagnostics in the clinical lab, and has potential for use in ecological studies.
Sun
Jiufeng
J
Department of Dermatology, The Second Affiliated Hospital, Sun Yat-Sen University, Guangzhou, China.
Li
Xiqing
X
Zeng
Hanxiang
H
Xie
Zhi
Z
Lu
Changming
C
Xi
Liyan
L
de Hoog
Gert S
GS
D000818:Animals; D001769:Blood / Q000382:microbiology; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D006801:Humans; D008168:Lung / Q000382:microbiology; D009181:Mycoses / Q000175:diagnosis* / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010407:Penicillium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D051381:Rats; D012867:Skin / Q000382:microbiology; D013045:Species Specificity; D013154:Spleen / Q000382:microbiology; D013997:Time Factors; D016610:Tissue Embedding / Q000379:methods*
D021903:DNA, Ribosomal Spacer