Abstract
Bordetella pertussis is an important human respiratory pathogen. Here, we describe a loop-mediated isothermal amplification (LAMP) method for the rapid detection of B. pertussis in clinical samples based on a visual test. The LAMP assay detected the BP485 target sequence within 60 min with a detection limit of 1.3 pg/μl, a 10-fold increase in sensitivity compared with conventional PCR. All 31 non-pertussis respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for B. pertussis. To evaluate the application of the LAMP assay to clinical diagnosis, of 105 sputum and nasopharyngeal samples collected from the patients with suspected respiratory infections in China, a total of 12 B. pertussis isolates were identified from 33 positive samples detected by LAMP-based surveillance targeting BP485. Strikingly, a 4.5 months old baby and her mother were found to be infected with B. pertussis at the same time. All isolates belonged to different B. pertussis multilocus sequence typing groups with different alleles of the virulence-related genes including four alleles of ptxA, six of prn, four of tcfA, two of fim2, and three of fim3. The diversity of B. pertussis carrying toxin genes in clinical strains indicates a rapid and continuing evolution of B. pertussis. This combined with its high prevalence will make it difficult to control. In conclusion, we have developed a visual detection LAMP assay, which could be a useful tool for rapid B. pertussis detection, especially in situations where resources are poor and in point-of-care tests.
Highlights
Bordetella pertussis, a Gram-negative bacterium first reported in 1906 [1], is an important human-specific respiratory pathogen that causes whooping cough in humans
OPTIMIZATION OF loop-mediated isothermal amplification (LAMP) ASSAY BP485 was targeted for detection of B. pertussis by a LAMP assay for the first time
SPECIFICITY OF LAMP ASSAY To evaluate the specificity of LAMP detection for B. pertussis, genomic DNA was extracted from B. pertussis ATCC18530, B. pertussis ATCC58003, B. pertussis ATCC53894, and 31 other pathogenic respiratory bacteria and tested using real-time turbidity or visual detection of color change as readouts
Summary
Bordetella pertussis, a Gram-negative bacterium first reported in 1906 [1], is an important human-specific respiratory pathogen that causes whooping cough (pertussis) in humans. It was revealed that neonates are commonly infected by adolescents and adults, before transmitting whooping cough further to infants and other groups. This change in transmission pattern from “child to child” to “adult to child” may be the major cause for the rising incidence of pertussis, especially in developed countries with high vaccination coverage [3,4,5,6,7]. Timely and accurate diagnosis is necessary for controlling the disease
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