Loop-mediated isothermal amplification assay for detection of Haemophilus influenzae type b in cerebrospinal fluid

Abstract

Haemophilus influenzae type b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection of bexA and a nested PCR for Hib (Hib PCR). No H. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.

Fig. 1.

(A) Nucleotide sequences of H. influenzae type b capsulation locus region II used to design the LAMP primer. The sequences used for LAMP primers are indicated by arrows. (B) Structure and sequence of the primers used in the LAMP reaction.

Fig. 2.

Electrophoretic analysis of the Hib LAMP-amplified products. Lane M, 100-bp ladder (New England Biolabs) used as a size marker; lane 1, LAMP product from lane 2 after digestion with Hpy188I (New England Biolabs) (the digested fragments were 111 and 152 bp); lane 2, 10⁶ copies of the genomic DNA of H. influenzae type b IID984; lane 3, no template.

Fig. 3.

(A) Detection limit and real-time reaction of the H. influenzae type b LAMP assay, as monitored by real-time measurements of turbidity. Shown from left to right are the curves of decreasing bacterial concentrations, from 1,000,000 to one genome copy. (B) The relationship between the threshold times (Tt) of each sample and the log of the amount of initial template DNA. The linear determination range was from 10 to 1,000,000 microorganisms per reaction mixture.