key: cord-0261822-49tmefit authors: Xu, H.; Chen, R.; Cai, Z.; Zhao, B.; Liu, X.; Liu, J. title: Universal two-dimensional labeled probe-mediated melting curve analysis based on multiplex PCR for rapid typing of plasmodium in a single closed tube date: 2022-04-16 journal: nan DOI: 10.1101/2022.04.12.22271963 sha: b5cef1bad0b082d9336e0b4655e1413691bb60e9 doc_id: 261822 cord_uid: 49tmefit Nowadays, malaria is still one of the major public health problems which commonly caused by four plasmodium species, especially in the epidemic of COVID-19 harboring similar symptoms of fever or fatigue, which easily result in misdiagnosis. The disadvantages of previous traditional detection methods, such as time-consuming, costly, complicated operation, strong professionalism, indistinguishable typing and so on, lead to the dilemma of difficulty to meet the clinical requirements of rapid, easy and accurate typing of common plasmodiums. Herein, we developed and maximally optimized a universal two-dimensional labeled probe-mediated melting curve analysis (UP-MCA) assay based on multiplex PCR for rapid and accurate typing of five plasmodiums, including novel human plasmodium, Plasmodium knowlesi (Pk), in a single closed tube following genome extraction. The assay showed the limit of detection (LOD) of 10 copies per reaction and can accurately distinguish plasmodium species from intra-plasmodium and other pathogens. In addition, we also proposed and verified different methods of fluorescence-quenching and two dimensional labeled tag for probes that are suitable for UP-MCA assay. Furthermore, its clinical performance was evaluated by 184 samples and showed sensitivity of 100% (164/164) and specificity of 100% (20/20) at 99% confidence interval, respectively, with the microscopy method as gold standard. Taken together, the UP-MCA system showed excellent sensitivity, specificity and accuracy for genotyping of plasmodium, and it meets the requirements of rapidity and convenience for plasmodium detection in clinical routine and has great potential for clinical translation. All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. detection have been implemented in some diagnostic laboratories as a supplement to 81 microscopy(5). Although they are rapid, simple, and easy to interpret, RDTs target 82 proteins specific to P. falciparum or P. vivax or those common to all plasmodium 83 species and cannot specifically differ P. malariae, P. ovale, and P. knowlesi(5, 6). 84 Furthermore, diagnostic sensitivity (e.g., lacking sensitivity for some strains of P. 85 falciparum and up to 50% of P. knowlesi)(7) and specificity (e.g., P. vivax in patients 86 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. including Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, 114 Plasmodium ovale, and Plasmodium knowlesi, with human ribonuclease P (RNase P) 115 gene as the internal control in state of one-pot and closed tube following introduction 116 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. annealing temperature (Tm) produced for hybridization with homology tag that 142 carried in plasmodium species-specific primer. Its process involves that polymerase 143 mediated asymmetric amplification using target specific primers for tag mark of 144 specific amplicon and fluorescent detection depending on hybridization between 145 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. Parameter replacement of annealing / extension at 64 ℃ and addition of 4 ng/uL ET 204 SSB were used for multiplex species-specific plasmodium detection assay. The 205 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. collected during the process of melting curve analysis, not for amplification process. The reaction was occurred by the fluorescence PCR equipment (Applied Biosystems 208 7500 Real-Time PCR System in this study; Thermo Fisher Scientific). (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. 254 UP-MCA detection assay is a method that uses asymmetric PCR to achieve the 255 enrichment of two-dimensional labelled products, and combines probe mediated 256 melting curve analysis technology to achieve specific detection of targets with 257 non-target sequence dependent universal fluorescent probes. With regards to 258 asymmetric PCR and melting curve analysis, the difference in the proportion of 259 forward and reverse primers will be introduced into amplification and 260 two-dimensional label should be integrated into amplicon, respectively. Moreover, to 261 further simplify primer design and minify optimization of assay for specificity 262 improvement, we adopted asymmetric PCR of plasmodium genus forward primer 263 which serves as abundant primer, and plasmodium specie-specific reverse primer with 264 homologous tag at the 5' end representing specific Tm value produced by 265 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. ; https://doi.org/10.1101/2022.04.12.22271963 doi: medRxiv preprint hybridization with universal probe, which serves as limiting primer for multiplex 266 UP-MCA plasmodium detection (Table 1) . These primers were based on target SSU 267 rRNA gene of plasmodium, which its copy number range from 4 to 8 and known to be 268 highly conserved regions suitable for molecular detection of human malaria parasites. This method was also helpful to improve the stability of Tm value of melting curve (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 297 To evaluate the sensitivity of the UP-MCA plasmodium detection assay, a series of 298 gradient concentration of diluted plasmids of plasmodium species and human gene 299 (RNase P, internal control) as reaction template were tested. The results showed that 325 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. (Table 2) . gene for the reason that not only that is known to be highly conserved regions suitable 354 for plasmodium-genus primers selection, but also its existence of plasmodium-species 355 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. Limitations were also demonstrated in the plasmodium UP-MCA detection assay. Firstly, there should be more targets to be validated for detection throughput 375 improvement used in plasmodium UP-MCA assay, and less detection objects and 376 numbers of primers may be the reason of ET SSB seldom seems to be functioned. But, 377 in other words, primers designed and used in the assay were adequately specific for 378 species-specific plasmodium. Secondly, the UP-MCA assay was one of 379 half-quantitative multiplex method that could not accurately achieve to quantitative 380 plasmodium species. Furthermore, more clinical samples collected from multicenter 381 should be incorporated into the assay for clinical performance validation. In summary, we developed one plasmodium species-specific detection assay for 383 greatly enhancing sensitivity and specificity that was important for import malaria 384 derived from regions or cities of frequent communications for economy or population 385 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 474 All rights reserved. No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted April 16, 2022. Plasmodium knowlesi: the fifth human malaria parasite A large focus of naturally acquired Plasmodium knowlesi infections in 398 human beings Guidelines for the Treatment of Malaria Human infections and detection of Plasmodium knowlesi Malaria rapid diagnostic tests Molecular diagnostic and surveillance tools for global malaria 404 control Evaluation of the 406 sensitivity of a pLDH-based and an aldolase-based rapid diagnostic test for diagnosis of 407 uncomplicated and severe malaria caused by PCR-confirmed Plasmodium knowlesi Plasmodium falciparum, and Plasmodium vivax Improved performance with saliva and urine 410 as alternative DNA sources for malaria diagnosis by mitochondrial DNA-based PCR assays An innovative tool 415 for moving malaria PCR detection of parasite reservoir into the field A novel 417 PCR-based system for the detection of four species of human malaria parasites and 418 Plasmodium knowlesi Detection of four 420 Plasmodium species in blood from humans by 18S rRNA gene subunit-based and 421 species-specific real-time PCR assays No reuse allowed without permission. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted Detection of Plasmodium knowlesi, Plasmodium falciparum and Plasmodium vivax using 427 loop-mediated isothermal amplification (LAMP) in a co-endemic area in Malaysia In 430 situ loop-mediated isothermal amplification (LAMP) for identification of Plasmodium species 431 in wide-range thin blood smears Multicopy targets for Plasmodium vivax and Plasmodium falciparum detection by 434 colorimetric LAMP Evaluation of loop-mediated isothermal amplification as a surveillance tool for 437 malaria in reactive case detection moving towards elimination Recombinase Polymerase 440 Amplification and Lateral Flow Assay for Ultrasensitive Detection of Low-Density Plasmodium 441 falciparum Infection from Controlled Human Malaria Infection Studies and Naturally Acquired 442 Infections Detection of Plasmodium knowlesi using recombinase polymerase 444 amplification (RPA) combined with SYBR Green I Recombinase Polymerase Amplification Combined with a Lateral 446 Flow Strip for the Detection of Plasmodium knowlesi Ultrasensitive CRISPR-based diagnostic for 449 field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria High-Throughput Two-Dimensional Polymerase Chain 452 Reaction Technology Multiplex 5' nuclease quantitative 454 real-time PCR for clinical diagnosis of malaria and species-level identification and 455 epidemiologic evaluation of malaria-causing parasites, including Plasmodium knowlesi Comparative evaluation of two 459 commercial real-time PCR kits (QuantiFast™ and abTES™) for the detection of Plasmodium 460 knowlesi and other Plasmodium species in Sabah, Malaysia Molecular detection of All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted April 16, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted April 16, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted April 16, 2022. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted April 16, 2022. ; https://doi.org/10.1101/2022.04.12.22271963 doi: medRxiv preprint