Development of a loop-mediated isothermal amplification assay for rapid detection of BK virus.
Loop-mediated isothermal amplification (LAMP) is a novel method for rapid amplification of DNA. Its advantages include rapidity and minimal equipment requirement. The LAMP assay was developed for BK virus (BKV), which is a leading cause of morbidity in renal transplant recipients. The characteristics of the assay, including its specificity and sensitivity, were evaluated. BKV LAMP was performed using various incubation times with a variety of specimens, including unprocessed urine and plasma samples. A ladder pattern on gel electrophoresis, typical of successful LAMP reactions, was observed specifically only for BKV and not for other viruses. The sensitivity of the assay with 1 h of incubation was 100 copies/tube of a cloned BKV fragment. Additionally, a positive reaction was visually ascertained by a simple color reaction using SYBR green dye. BKV LAMP was also successful for urine and plasma specimens without the need for DNA extraction. Due to its simplicity and specificity, the LAMP assay can potentially be developed for "point of care" screening of BKV.
Bista
Bipin Raj
BR
Department of Pediatrics, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA.
Ishwad
Chandra
C
Wadowsky
Robert M
RM
Manna
Pradip
P
Randhawa
Parmjeet Singh
PS
Gupta
Gaurav
G
Adhikari
Meena
M
Tyagi
Rakhi
R
Gasper
Gina
G
Vats
Abhay
A
D001739:BK Virus / Q000302:isolation & purification*; D001483:Base Sequence; D004279:DNA, Viral; D005814:Genes, Viral; D006801:Humans; D016030:Kidney Transplantation / Q000009:adverse effects; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D027601:Polyomavirus Infections / Q000175:diagnosis / Q000821:virology; D012680:Sensitivity and Specificity; D013997:Time Factors; D014412:Tumor Virus Infections / Q000175:diagnosis / Q000821:virology
D004279:DNA, Viral