key: cord-0991649-4ocnnq83
authors: Kitagawa, Masae; Nagamine, Kentaro; Oka, Hiroko; Ouhara, Kazuhisa; Ogawa, Ikuko; Komatsuzawa, Hitoshi; Kurihara, Hidemi
title: Rapid detection of the Streptococcus mutans cnm gene by loop-mediated isothermal amplification
date: 2020-06-25
journal: Anal Biochem
DOI: 10.1016/j.ab.2020.113812
sha: 0be2523b4549e32afac4bac0ef24f1d89822d9f2
doc_id: 991649
cord_uid: 4ocnnq83

This study investigated a method using loop-mediated isothermal amplification (LAMP) for the rapid detection of cnm-positive Streptococcus mutans (S. mutans) associated with cerebral microhemorrhage. LAMP amplified the cnm gene plasmid vector, but not human or microbial genomic DNA. The cnm DNA of the cnm-positive S. mutans strain was detected in saliva without DNA extraction after 1 day of culture. This method resulted in a cnm-positive rate of 26.4% in 102 samples, which was higher than that obtained with conventional PCR. In conclusion, LAMP may be used for the detection of cnm-positive S. mutans in a large number of samples.

Streptococcus mutans (S. mutans) is a pathogen that causes dental 43 caries, which may result in tooth loss. Recent studies have reported that S. 44 mutans is associated with systemic diseases in addition to periodontal disease 45 [1, 2] . Specifically, S. mutans with cnm collagen-binding ability is associated with 46 cerebral microbleeds [3, 4] . Furthermore, infection with cnm-positive S. mutans 47 is a potential risk factor for cerebral hemorrhagic stroke [5] . Therefore, it is 48 important to evaluate whether a patient is infected with cnm-positive S. mutans. 49

The conventional method for detecting S. mutans DNA is to culture saliva 50 or dental plaque on Mitis-Salivarius agar containing bacitracin (MSB) for 48 h. 51

Colonies are subsequently incubated in brain heart infusion broth (BHI) for 1 day. 52 DNA is extracted from the bacteria, and the target S. mutans gene is amplified 53 by the polymerase chain reaction (PCR) [3] . This time-consuming and 54 labor-intensive process typically takes up to 3-4 days to complete. 55

The loop-mediated isothermal amplification (LAMP) method was 56 developed to ensure the specific, efficient, and rapid amplification of DNA under 57 isothermal conditions [6-8]. Furthermore, loop primers, which hybridize to the 58 stem-loops, and prime strand displacement DNA synthesis reduce the reaction 59 time to less than half that of the original LAMP method [9] . Additionally, calcein, 60 the main component of the fluorescence visual detection reagent, binds 61 manganese ions that quench fluorescence. Thus, when a manganese ion is 62 deprived of the pyrophosphate ion generated by LAMP, fluorescence is emitted [10] . The LAMP method can be applied for the direct detection of pathogens in 64 crude samples without DNA purification steps [11, 12] , and has been used to 65 detect the causative agents of tuberculosis, mycoplasma, influenza, and severe 66 acute respiratory syndrome [13] [14] [15] [16] . The rapid detection of causative bacteria 67 and viruses leads to prompt treatment and prevention, even in chronic diseases. 68

The present study investigated a method using LAMP for the rapid detection of 69 cnm-positive S. mutans associated with cerebral microhemorrhage. 70

All experimental protocols were approved by the epidemiological research ethics 72 review committee of Hiroshima University (no. E-786-2). All subjects provided 73 written informed consent. 74

Vector construction: The S. mutans cnm gene fragment was amplified from a 75 PCR product using the Expand Long Template kit (Roche, Toyo, Japan), as 76 previously described [3] , and cloned into pGEM-T Easy (Promega, Tokyo, 77 Japan), a thymidine adenine cloning vector. The resulting product was named 78 pGEMcnm. The primer set used for the cnm gene was as follows: forward primer 79 Specificity and sensitivity test: To examine specificity, the LAMP reaction 106 was performed using human genomic DNA and the pGEMcnm plasmid vector 107 as templates. The pGEMcnm plasmid vector was amplified by LAMP, but human 108 genomic DNA was not (Fig. 1A ). This suggested that the primer set used did not 109 amplify DNA derived from human oral cells in the salivary samples. In the 110 sensitivity test, cnm was detected with 10 3 pGEMcnm plasmid vector molecules, 111

but not with 10 2 molecules (Fig. 1B) . In the diluted solution of cnm-positive 112 strains, cnm was detected with 10 -5 OD 600 , but not with 10 -6 OD 600 , suggesting 113 that the LAMP method may be used to detect the cnm gene if ~10 3 bacterial 114 cells are present (Fig. 1C) . For detection in a fewer number of cells, direct 115 detection in saliva may still be possible though judicious primer design. S. 116 sobrinus, P. gingivalis, and Candida spp., which are present in the oral cavity of 117 several mammals, do not have the cnm gene and were not detected (Fig. 1D) . 118 Therefore, the novel method described in the present study can accurately 119 detect cnm. 120

Comparison of culture conditions: Saliva containing cnm-positive S. 121 mutans was cultured on MSB agar, and LAMP was subsequently performed. A 122 1-day culture in MSB agar is required to selectively culture S. mutans, because 123 one milliliter of human saliva from a healthy adult contains approximately 100 124 million bacterial cells [17] . The results showed that cnm DNA was detected 125 without DNA extraction after 1 day of culture. However, when the same saliva was cultured in BHI medium alone, cnm DNA was not detected, even after 1-2 days of growth (Fig. 1E) . This result suggested that LAMP enabled the detection 128 of cnm following a short culture period, even when DNA was not extracted. mutans on MSB agar. BHI was used to collect the bacterial colonies; however, 143 tris-ethylenediaminetetraacetic acid buffer or distilled water may also be used. 144

Since cnm-positive S. mutans can be directly detected using the liquid, the 145 results can be obtained within 2 days (Fig. 2) . Thus, LAMP appears to be 146 effective in screening cnm-positive S. mutans carriers and these patients can be 147 forewarned of the risks for cerebral microbleeds and cognitive decline. Early detection enables proactive treatment, thereby diminishing the risk of 149 deterioration in quality of life. 150

We would like to thank Dr. Shrestha Madhu (Graduate School of Biomedical and 153

Health Sciences, Hiroshima University) for editing assistance. 154

This work was supported by JSPS KAKENHI grant number JP20K02352 (to 157

Kentaro Nagamine) and JP20K09940 (to Masae Kitagawa). 158 Primers used in the present study 

A novel and rapid 201 loop-mediated isothermal amplification assay for the specific detection of 202

An integrated direct 204 loop-mediated isothermal amplification microdevice incorporated with an 205 immunochromatographic strip for bacteria detection in human whole blood 206 and milk without a sample preparation step

Operational 210 feasibility of using loop-mediated isothermal amplification for diagnosis of 211 pulmonary tuberculosis in microscopy centers of developing countries

Detection of Mycoplasma 215 pneumoniae by loop-mediated isothermal amplification (LAMP) assay and 216 serology in pediatric community-acquired pneumonia

Development and evaluation of a novel loop-mediated 220 isothermal amplification method for rapid detection of severe acute

Evaluation of reverse transcription 224 loop-mediated isothermal amplification assays for rapid diagnosis of 225 pandemic influenza A/H1N1 2009 virus

The relationship of the oral 227 microbiotia to periodontal health and disease

Molecular characterization of Streptococcus mutans strains 232 containing the cnm gene encoding a collagen-binding adhesin

Streptococcus 236 mutans clonal variation revealed by multilocus sequence typing

Molecular and clinical analyses of the 240 gene encoding the collagen-binding adhesin of Streptococcus mutans

Easy detection of cnm gene related to cerebral hemorrhage

Non-invasive sampling using saliva

Reduced detection time due to shorter culture period and not extracting DNA

The study was conducted and designed by Masae Kitagawa, Kentaro Nagamine,