Development and evaluation of loop-mediated isothermal amplification assay for rapid and inexpensive detection of cytomegalovirus DNA in vitreous specimens from suspected cases of viral retinitis

Abstract

A loop-mediated isothermal amplification (LAMP) assay for the rapid detection of cytomegalovirus (CMV) was developed and evaluated. The LAMP assay specifically amplified only CMV DNA, and no cross-reactivity with the DNA of herpes simplex virus type 1, varicella-zoster virus, adenovirus, Aspergillus flavus, or Staphylococcus aureus was observed. The sequences of the LAMP assay-positive CMV products were perfectly (100%) matched with the CMV sequence deposited in the GenBank database. The sensitivity of the LAMP assay was found to be 10 copies/microl of CMV DNA. Vitreous samples from 40 patients with suspected retinitis were subjected to LAMP and real-time PCR for the detection of CMV. Of 40 patients with suspected viral retinitis, 10 tested positive for CMV by the real-time PCR and LAMP assays. A 100% concordance was observed between the results of the two methods. The LAMP assay is a rapid, highly specific, and sensitive method for the diagnosis of retinitis caused by CMV.

FIG. 1.

Oligonucleotide primers used for LAMP assay of cytomegalovirus (GenBank accession no. U66425) within the gB gene. F1c and B1c, sequences complementary to the F1 and B1 regions, respectively; arrows, orientations of the various primers used in the CMV LAMP assay.

FIG. 2.

Specificity of LAMP assay. Lane 1, 100-bp DNA ladder; lane 2, negative control; lanes 3 and 4, CMV, for which LAMP assay products were obtained; lanes 5, 6, 7, 8, and 9, other organisms (HSV-1, VZV, adenovirus, Aspergillus flavus, and Staphylococcus aureus, respectively), for which no LAMP products were obtained. The products amplified by the LAMP assay exhibit a typical ladder-like pattern on electrophoresis, indicating the formation of stem-loop DNA with inverted repeats.

FIG. 3.

The tube with a positive reaction (tube 2) shows a color change to yellowish green, which can be distinguished from the reddish orange color of a negative reaction (tube 1).

FIG. 4.

Sensitivity of LAMP assay. Lane 1, 100-bp DNA ladder; lane 2, negative control; lane 3, 10,000 copies/μl of CMV DNA; lane 4, 1,000 copies/μl of CMV DNA; lane 5, 100 copies/μl of CMV DNA; lane 6, 10 copies/μl of CMV DNA; lane 7, 10-fold dilution of 10 copies/μl of CMV DNA.