Capture and Ligation Probe-PCR (CLIP-PCR) for Molecular Screening, with Application to Active Malaria Surveillance for Elimination

Cheng, Zhibin; Wang, Duoquan; Tian, Xiaoyi; Sun, Yu; Sun, Xin; Xiao, Ning; Zheng, Zhi

doi:10.1373/clinchem.2014.237115

Cited by 23 publications

(24 citation statements)

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“…The standard curve was made by threefold serial dilutions (8–0.004 parasites/µL) of a lysate of cultured P. falciparum 3D7 strain diluted with a parasite-negative whole blood lysate. For CLIP-PCR, the sample was considered positive if the fluorescent signal increased within 29 cycles and the melting curve was the same as that of the positive control [36]. …”

Section: Methodsmentioning

confidence: 99%

“…Several different target genes have been used, including the 18S ribosomal RNA gene ( 18S rRNA ) [15–19], tRNA [20], AMA1 [21], and cytochrome b [22, 23], among which the 18S rRNA gene is the most commonly used [11, 24–26]. PCR-based methods include nested PCR with DNA (nD-PCR) [12, 24, 27–31], nested reverse transcriptase PCR (nRT-PCR) [26, 30], quantitative RT-PCR [27, 32–35], and more recently, capture and ligation probe-PCR (CLIP-PCR) [36]. PCR can typically detect 5–10 parasites/µL, while nRT-PCR can detect as few as 22 parasites/mL [26].…”

Section: Introductionmentioning

confidence: 99%

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Comparison of methods for detecting asymptomatic malaria infections in the China–Myanmar border area

Zhao

et al. 2017

Malar J

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BackgroundSensitive methods for detecting asymptomatic malaria infections are essential for identifying potential transmission reservoirs and obtaining an accurate assessment of malaria epidemiology in low-endemicity areas aiming to eliminate malaria. PCR techniques to detect parasite nucleic acids (DNA or RNA) are among the most commonly used molecular methods. However, most of these methods are of low throughput and cannot be used for large-scale molecular epidemiological studies. A recently developed capture and ligation probe-PCR (CLIP-PCR) is claimed to have the sensitivity of molecular techniques and the high throughput capacity needed for screening purposes. This study aimed to compare several molecular methods for detecting asymptomatic and submicroscopic Plasmodium infections in healthy residents of a malaria-hypoendemic region in Southeast Asia, where malaria elimination is in sight.MethodThis study compared three molecular detection methods side-by-side, namely nested PCR targeting the rRNA genes, nested RT-PCR to detect parasite rRNA, and CLIP-PCR to detect parasite rRNA in 1005 healthy individuals in northeastern Myanmar. For nested PCR and RT-PCR, parasite DNA and total RNA were extracted from ~100 µL of blood, whereas RNA used for CLIP-PCR was from a 3 mm disk of dried blood filter paper. The sensitivity and specificity of these methods were compared with those of conventional light microscopy. In addition, RT-PCR and quantitative RT-PCR (qRT-PCR) targeting the Pvs25 gene in Plasmodium vivax were used to assess gametocyte prevalence in the samples.ResultsLight microscopy detected Plasmodium infections in only 1.19% of the residents harbouring the parasites. CLIP-PCR had slightly better performance and detected Plasmodium infections in 1.89% of the population. Further improvement was achieved by nested PCR to detect parasite DNA, which detected P. vivax and Plasmodium falciparum infections in 2.39% of the residents. The nested RT-PCR targeting rRNA, however, detected as many as 187 (18.61%) individuals having Plasmodium infections with P. vivax being the predominant species (176 P. vivax, 5 P. falciparum and 6 P. falciparum/P. vivax mixed infections). Of the 210 Plasmodium-positive samples detected by all molecular methods, 115 were Pvs25-positive by qRT-PCR, indicating that a large proportion of asymptomatic individuals were gametocyte carriers.ConclusionNested RT-PCR based on the detection of asexual-stage parasite rRNA was the most sensitive, with a more than sixfold higher sensitivity than the other two molecular methods of parasite detection. CLIP-PCR has an increased throughput, but its sensitivity in this study was much lower than those of other molecular methods, which may be partially due to the smaller amount of RNA input used.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-017-1813-0) contains supplementary material, which is available to authorized users.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Comparison of methods for detecting asymptomatic malaria infections in the China–Myanmar border area

Zhao

et al. 2017

Malar J

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show abstract

“…Consistent with other nucleic acid–based tests that target 18S rRNA, CLIP-PCR applied by Cheng et al may benefit from the parasites amplifying the target sequence themselves (1). Additionally, there is possibility that the methods used by Cheng et al to capture 18S rRNA, which are reported to avoid DNA extraction, may be highly efficient and contribute to the reported superior evaluation of pooled dried blood spot samples.…”

Section: Strengths and Limitations Of Clip-pcrmentioning

confidence: 79%

“…Additionally, there is possibility that the methods used by Cheng et al to capture 18S rRNA, which are reported to avoid DNA extraction, may be highly efficient and contribute to the reported superior evaluation of pooled dried blood spot samples. Specifically, in their study, Cheng et al performed tests on serial dilutions of an in vitro culture of P. falciparum (laboratory-adapted strain 3D7) showing that their LOD was 0.01 parasitized cells per microliter of blood (1). Furthermore, they reported not seeing any reduced capacity to detect their P. falciparum target sequence even in pools of up to 26 samples that might dilute nucleic acid concentration when combined with uninfected samples.…”

Section: Strengths and Limitations Of Clip-pcrmentioning

confidence: 99%

“…In the current issue of Clinical Chemistry , Cheng et al describe their application of a recently developed commercial kit to perform molecular diagnosis of malaria by a capture and ligation probe-PCR (CLIP-PCR) 2 strategy (1). Some review of the efforts directed against malaria is necessary to understand how CLIP-PCR will integrate into the current global malaria elimination effort.…”

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confidence: 99%

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