A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay

Sakai, Jun; Maeda, Takeshi; Tarumoto, Norihito; Misawa, Kazuhisa; Tamura, Shinsuke; Imai, K.; Yamaguchi, Toshiyuki; Iwata, Satoshi; Murakami, Takashi; Maesaki, Shigefumi

doi:10.1016/j.mimet.2017.08.009

Cited by 11 publications

(18 citation statements)

References 37 publications

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“…However, complete suppression of non-target alleles was difficult (Additional file 3 (h) LAMP_ms1-2). The use of PNA (peptide nucleic acid) [ 15 ] to block the alternative allele and to increase the specificity (PNA-LAMP) is an avenue for further study to better refine this method (Additional file 7 ).…”

Section: Discussionmentioning

confidence: 99%

“… Multiplex PCR may need optimization, depending on markers (such as microsatellites) combined and PCR machines used. LAMP assays may be further optimized by using other primer combinations or applying other methods, such as PNA-LAMP [ 15 ]. Sensitivity and specificity of LAMP assay has not been determined, and using both positive and negative controls is necessary to cross-validate the assay.…”

Section: Limitationsmentioning

confidence: 99%

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Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don

Ueno

Wei

et al. 2020

BMC Res Notes

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Objective Due to the allergic nature of the pollen of Cryptomeria japonica, the most important Japanese forestry conifer, a pollen-free cultivar is preferred. Mutant trees detected in nature have been used to produce a pollen-free cultivar. In order to reduce the time and cost needed for production and breeding, we aimed to develop simple diagnostic molecular markers for mutant alleles of the causative gene MALE STERILITY 1 (MS1) in C. japonica to rapidly identify pollen-free mutants. Results We developed PCR and LAMP markers to detect mutant alleles and to present experimental options depending on available laboratory equipment. LAMP markers were developed for field stations, where PCR machines are unavailable. The LAMP method only needs heat-blocks or a water bath to perform the isothermal amplification and assay results can be read by the naked eye. Because the causative mutations were deletions, we developed two kinds of PCR markers, amplified length polymorphism (ALP) and allele specific PCR (ASP) markers. These assays can be visualized using capillary or agarose gel electrophoresis.

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Section: Discussionmentioning

confidence: 99%

Section: Limitationsmentioning

confidence: 99%

Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don

Ueno

Wei

et al. 2020

BMC Res Notes

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“…It can be seen that higher cell uptake rate and slower cell clearance rate are specific manifestations of antisense peptide nucleic acid in K1 cells, which support the conclusion that antisense PNA can be used to modulate the transcription and translation of tumor genes, and that their high stability and strong binding affinity make these molecules useful for detecting tumor cells. 16,34 Preclinical studies of 68 GA-labeled ODNs for pulmonary malignancies have been designed to target the activated human K-ras oncogene; 35 an increasing number of prospective studies have begun to apply antisense therapy using 125 I or 111 In in patients with central nervous system and cervical cancers. [36][37][38] This study once again confirmed the feasibility of antisense imaging and further explored the efficacy of antisense therapy using 177 Lu coupling, which is based on study showed that PNA can be metabolized in cells over several months.…”

Section: Discussionmentioning

confidence: 99%

“…15 Peptide nucleic acid (PNA) was developed from oligonucleotides (ODN), which can block the function of RNA polymerase structurally and thus interferes with gene transcription and downregulates the expression of target genes. 16 Compared to ODN, PNA has the characteristics of easy absorption, strong specificity and high stability, which may facilitate the treatment of tumors and blood diseases. Therefore, blocking CITED1 expression by an antisense PNA (asPNA) binding CITED1 mRNA may be one of the effective methods for the diagnosis and treatment of iodine resistance to PTC.…”

Section: Introductionmentioning

confidence: 99%

<p>Preparation and SPECT/CT Imaging of <sup>177</sup>Lu-Labeled Peptide Nucleic Acid (PNA) Targeting CITED1: Therapeutic Evaluation in Tumor-Bearing Nude Mice</p>

Li¹,

Zhang²,

Li³

et al. 2020

OTT

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The expression of Cbp/p300-interacting transactivator with Glu/Asp-rich carboxyterminal domain 1 (CITED1) is upregulated in papillary thyroid carcinoma (PTC) and mediates cell proliferation and migration. To facilitate early diagnosis and monitoring of recurrent or metastatic PTC, we designed 177 Lu-labeled antisense peptide nucleic acid (PNA) targeting CITED1 mRNA to evaluate the therapeutic potential, while analyzing its distribution in nude mice and the characteristics withsingle-photon emission-computed tomography/ computed tomography (SPECT/CT) imaging. Materials and Methods: 177 Lu-DOTA-anti-CITED1-PNA (177 Lu-asPNA) was obtained by indirect labeling. High-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC) were used to determine the labeling rate and radiochemical purity. The stability of 177 Lu-asPNA was evaluated by TLC, and the radioactivity count was measured by a γ counter to calculate its uptake capacity in K1 cells. To analyze the distribution of 177 Lu-asPNA in body tissues and organs of nude mice, static single-photon emission-computed tomography (SPECT) imaging and SPECT/CT image fusion were performed. Then, the therapeutic effects of probes were explored by tumor growth curves and survival analysis. Results: Our probe showed a radiochemical purity of 96.5±0.15% at 1 hr and specific activity of 8.7±0.53 MBq/μg. The uptake rate in the 177 Lu-asPNA group was much higher than that in the 177 Lu-DOTA-nonsense-PNA (177 Lu-nonsense-PNA) and 177 Lu-DOTA groups (P<0.05). The biological distribution showed that the tumor/muscle ratio was at its highest at 24 h (4.98±0.34) post-inoculation, with SPECT/CT imaging showing clear tumor development. By measuring tumor volume of tumor-bearing nude mice, the 177 Lu-asPNA group showed a significant difference in tumor size 9 days after injection (P < 0.05). Kaplan-Meier survival curves showed that the overall survival rate in the 177 Lu-asPNA group was significantly different from those in the DOTA-anti-CITED1-PNA (asPNA) and saline groups (P = 0.002, log-rank test). Conclusion: 177 Lu-asPNA was developed successfully, showing a high labeling rate and good stability. SPECT/CT imaging demonstrated tumor growth in nude mice, which was effectively inhibited by our probe, thus prolonging survival.

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“…Loopamp Realtime Turbidimeter LA-200, LA-320, LA-500, Eiken Chemical Co., Ltd., Tokyo, Japan) is commonly used for LAMP product detection. LAMP assay was monitored for the white precipitation caused by the presence of magnesium pyrophosphate, Mg 2 P 2 O 7 (a by-product of LAMP reaction) at optical density 650 nm every 6 s. Several studies have been reported using turbidity to detect the presence of desired micro-organisms, for instances, Mycoplasma pneumoniae (Sakai et al 2017), Salmonella spp. (Mashooq et al 2016), human papillomavirus (Kumvongpin et al 2016) and Clostridium difficile (Lin et al 2015).…”

Section: Lamp End-point Detection Methodsmentioning

confidence: 99%

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

et al. 2018

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A novel detection procedure for mutations in the 23S rRNA gene of Mycoplasma pneumoniae with peptide nucleic acid-mediated loop-mediated isothermal amplification assay

Cited by 11 publications

References 37 publications

Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don

Development of diagnostic PCR and LAMP markers for MALE STERILITY 1 (MS1) in Cryptomeria japonica D. Don

<p>Preparation and SPECT/CT Imaging of <sup>177</sup>Lu-Labeled Peptide Nucleic Acid (PNA) Targeting CITED1: Therapeutic Evaluation in Tumor-Bearing Nude Mice</p>

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

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