Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria

被引：40

作者：

Chua, Kek Heng ^{[1
]}

Lim, Siew Chee ^{[2
]}

Ng, Ching Ching ^{[2
]}

Lee, Ping Chin ^{[3
]}

Lim, Yvonne Ai Lian ^{[4
]}

Lau, Tze Pheng ^{[5
]}

Chai, Hwa Chia ^{[1
]}

机构：

[1] Univ Malaya, Fac Med, Dept Biomed Sci, Kuala Lumpur 50603, Malaysia

[2] Univ Malaya, Fac Sci, Inst Biol Sci, Kuala Lumpur 50603, Malaysia

[3] Univ Malaysia Sabah, Sch Sci & Technol, Kota Kinabalu 88400, Sabah, Malaysia

[4] Univ Malaya, Fac Med, Dept Parasitol, Kuala Lumpur 50603, Malaysia

[5] Univ Malaya, Dept Pathol, Fac Med, Kuala Lumpur 50603, Malaysia

来源：

SCIENTIFIC REPORTS | 2015年 / 5卷

关键词：

REAL-TIME PCR; POLYMERASE-CHAIN-REACTION; SYBR-GREEN-I; PLASMODIUM-VIVAX; LABORATORY DIAGNOSIS; CONVENTIONAL PCR; NESTED-PCR; BLOOD; ASSAY; IDENTIFICATION;

D O I：

10.1038/srep15671

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNex (TM), a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1-100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNex (TM). This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNex (TM). The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis.

引用

页数：13

共 59 条

[1]

[Anonymous], T R SOC TROP MED S1

[2] PCR-based detection of Plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods [J].

Bass, Chris ;

Nikou, Dimitra ;

Blagborough, Andrew M. ;

Vontas, John ;

Sinden, Robert E. ;

Williamson, Martin S. ;

Field, Linda M. .

MALARIA JOURNAL, 2008, 7 (1)

[3] Quantitative real-time PCR is not more sensitive than "Conventional" PCR [J].

Bastien, Patrick ;

Procop, Gary W. ;

Reischl, Udo .

JOURNAL OF CLINICAL MICROBIOLOGY, 2008, 46 (06) :1897-1900

[4]

Benson DA, 2013, NUCLEIC ACIDS RES, V41, pD36, DOI [10.1093/nar/gkn723, 10.1093/nar/gkp1024, 10.1093/nar/gkw1070, 10.1093/nar/gkr1202, 10.1093/nar/gkx1094, 10.1093/nar/gkl986, 10.1093/nar/gkq1079, 10.1093/nar/gks1195, 10.1093/nar/gkg057]

[5] Comparison of three molecular methods for the detection and speciation of Plasmodium vivax and Plasmodium falciparum [J].

Boonma, Prapaporn ;

Christensen, Peter R. ;

Suwanarusk, Rossarin ;

Price, Ric N. ;

Russell, Bruce ;

Lek-Uthai, Usa .

MALARIA JOURNAL, 2007, 6 (1)

[6] DEMONSTRATION BY THE POLYMERASE CHAIN-REACTION OF MIXED PLASMODIUM-FALCIPARUM AND PLASMODIUM-VIVAX INFECTIONS UNDETECTED BY CONVENTIONAL MICROSCOPY [J].

BROWN, AE ;

KAIN, KC ;

PIPITHKUL, J ;

WEBSTER, HK .

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, 1992, 86 (06) :609-612

[7] Hexaplex PCR Detection System for Identification of Five Human Plasmodium Species with an Internal Control [J].

Chew, Ching Hoong ;

Lim, Yvonne Ai Lian ;

Lee, Ping Chin ;

Mahmud, Rohela ;

Chua, Kek Heng .

JOURNAL OF CLINICAL MICROBIOLOGY, 2012, 50 (12) :4012-4019

[8] Increased sensitivity of malaria detection by nested polymerase chain reaction using simple sampling and DNA extraction [J].

Cox-Singh, J ;

Mahayet, S ;

Abdullah, MS ;

Singh, B .

INTERNATIONAL JOURNAL FOR PARASITOLOGY, 1997, 27 (12) :1575-1577

[9] Simultaneous identification of the four human Plasmodium species and quantification of Plasmodium DNA load in human blood by real-time polymerase chain reaction [J].

de Monbrison, F ;

Angei, C ;

Staal, A ;

Kaiser, K ;

Picot, S .

TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE, 2003, 97 (04) :387-390

[10] A TaqMan real-time PCR assay for the detection and quantitation of Plasmodium knowlesi [J].

Divis, Paul C. S. ;

Shokoples, Sandra E. ;

Singh, Balbir ;

Yanow, Stephanie K. .

MALARIA JOURNAL, 2010, 9

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