Development of a single-tube loop-mediated isothermal amplification assay for detection of four pathogens of bacterial meningitis

Abstract

Several loop-mediated isothermal amplification (LAMP) assays have been developed to detect common causative pathogens of bacterial meningitis (BM). However, no LAMP assay is reported to detect Streptococcus agalactiae and Streptococcus suis, which are also among common pathogens of BM. Moreover, it is laborious and expensive by performing multiple reactions for each sample to detect bacterial pathogen. Thus, we aimed to design and develop a single-tube LAMP assay capable of detecting multiple bacterial species, based on the nucleotide sequences of the 16S rRNA genes of the bacteria. The nucleotide sequences of the 16S rRNA genes of main pathogens involved in BM were aligned to identify conserved regions, which were further used to design broad range specific LAMP assay primers. We successfully designed a set of broad range specific LAMP assay primers for simultaneous detection of four species including Staphylococcus aureus, Streptococcus pneumoniae, S. suis and S. agalactiae. The broad range LAMP assay was highly specific without cross-reactivity with other bacteria including Haemophilus influenzae, Neisseria meningitidis and Escherichia coli. The sensitivity of our LAMP assay was 100-1000 times higher compared with the conventional PCR assay. The bacterial species could be identified after digestion of the LAMP products with restriction endonuclease DdeI and HaeIII.

Development of loop-mediated isothermal amplification to detect Streptococcus suis and its application to retail pork meat in Japan

“…Therefore, we considered LAMP to be a useful tool to detect S. suis contamination in foods. Huy et al (2012) designed primers according to the sequences of the 16S rRNA genes common to four bacterial species (Staphylococcus aureus, Streptococcus pneumoniae, S. suis, and Streptococcus agalactiae) and established a single-tube LAMP technique. However, it requires a 90-min procedure for enzyme digestion of the amplified product and two rounds of gel electrophoresis to identify bacterial species, and also cannot determine the bacterial serotypes.…”

A paper/polymer hybrid CD-like microfluidic SpinChip integrated with DNA-functionalized graphene oxide nanosensors for multiplex qLAMP detection

“…3 Multiplex LAMP can amplify several DNA targets in one reaction and be employed for simultaneous detection of multiple pathogens, which can provide richer information and more conveniences to end users, consume fewer samples and reagents, and provide faster diagnosis to the patient. 4 The mLAMP detection is challenging mainly due to complexities from multiple primer sets and complicated ladder-pattered LAMP products from loop-mediated reactions. As a result, the traditional tube-based mLAMP can only identify the occurrence of amplification but cannot effectively identify the specific pathogen.…”

Rapid Visual Detection of Highly Pathogenic Streptococcus suis Serotype 2 Isolates by Use of Loop-Mediated Isothermal Amplification

“…Huy et al (17) designed primers according to the common 16S rRNA sequences of four bacterial species (Staphylococcus aureus, Streptococcus pneumoniae, S. suis, and Streptococcus agalactiae) and established a single-tube LAMP technique to amplify bacterial nucleic acid. However, this method requires a 90-min period for enzyme digestion of the amplified product and two rounds of gel electrophoresis to identify bacterial species, and it cannot distinguish between bacterial serotypes.…”