关键词: 疟原虫, 染料法qPCR, 一步反转录qPCR, 最低检出限

Abstract:

Objective To establish a qPCR method for detecting the 18S rDNA of the asexual stage (stage A) of 5 Plasmodium species and to evaluate its detection efficacy. Methods The 18S rDNA sequences of stage A, sporozoite stage （stage S） and oocyst (stage O) of five human malaria parasite species and the sequence from human were downloaded from GenBank and aligned to confirm the distribution pattern of the conserved and variable regions. Specific primers of Plasmodium genus were designed in the conserved regions and the specific primers of species were designed in the variable regions. Specific primer pairs were screened by the qPCR amplifying the DNA of the five Plasmodium species, Microtus babesi and Leishmania infantis as templates, and the optimal annealing extension temperature and primer concentration of the specific primer pairs were screened. The plasmids of short 18S rDNA fragments (amplified with screened primers) and long 18S rDNA fragments (amplified DNA fragments with conserved primers) of the five Plasmodium species of stage A were constructed respectively. The qPCR amplification was carried out with plasmid DNA [(5 × 10¹） - (5 × 10⁸) copy/μl] of the short fragments and plasmid DNA [(5 × 10⁴）- (5 × 10⁹) copy/μl] of the long fragment as templates, and the amplification efficiency, the minimum detection limit [including the minimum plasmid concentration and the corresponding cycle threshold (Ct) value, the coefficient of variation of repeated detection], the maximum concentration of non-target DNA when there was no amplification, and the maximum copy concentration ratio between the different Plasmodium species in the detectable mixed DNA were determined respectively. Using the DNA of the 18 blood samples infected with Plasmodium parasites (6 samples of P. falciparum, 6 samples of P. vivax, 2 samples of P. malariae, 4 samples of P. ovale, and 1 sample of P. knowlesi), which were diluted by 5 folds as the template, the selected specific primers were used for qPCR and one-step reverse transcription qPCR amplification respectively, and nested PCR amplification was performed at the same time to calculate and compare the density of the samples at the lowest detection limit. Using selected specific primers, qPCR amplification was performed on the DNA from 70 malaria patients’ blood samples (26 samples of P. falciparum, 14 samples of P. vivax, 14 samples of P. malariae, 9 samples of P. ovale, 3 samples of P. knowlesi, 4 samples of mixed infections), 65 whole blood samples of malaria-negative patients, and Amoeba sp. (1 sample), M. babesi (1 sample), and Leishmania donovani (3 samples) samples to calculate the sensitivity, specificity and conformity. Chi-square test, t-test, Mann-Whitney test or Wilcoxon rank sum test were used to compare the results of different PCR methods. Results Sequence alignment results showed that the consistency of the 18S rDNA sequence of the five Plasmodium species of parasites in stage A was 89.3%-96.7%, higher than those in stage S (60.0%-92.1%). Nine conserved regions and eight variable regions were mapped. Except for the 18S rDNA fragments of stage O of P. vivax and stage S of P. malraiae, the sequence length of the conserved regions was the same with consistency of ≥ 92%. One pair of generic primers and 5 pairs of specific species primers were selected for detecting the five species of human malaria parasites. When amplified with qPCR, the amplification efficiency of all primer pairs were 90.6%-98.8%, the optimal annealing extension temperature was 57.8 ℃, and the optimal final primer concentration was 0.3 μmol/L. The minimum detection limit for the short fragment plasmid was 5 × 10² copies/μl, the corresponding Ct value is 32.3-33.1 and the coefficient of variation of repeated detection is 0.5%-3.7%. The highest concentration of non-target DNA was (5 × 10⁶)-(5 × 10⁸) copies/μl when no amplification presented. The maximum copy concentration ratio between different Plamodium species in the mixed DNA that can be detected reached 10⁴. The average minimum paparsite density detected by one-step reverse transcription qPCR, qPCR and nested PCR were (4.1 ± 5.1), (6.0 ± 4.3) and (8.2 ± 2.9) parasites/μl respectively, and the lowest detected protozoa density was 0.01, 0.3 and 2.0 parasites/μl respectively, and the coefficient of variation were 125.6%, 72.2% and 35.8%, respectively. The sensitivity of the one-step reverse transcription qPCR is higher than those of the other two methods (Wilcoxon rank sum test, Z = -2.0, -2.0; P < 0.05). There was no statistically significant difference in the sensitivity between qPCR and nested PCR (t = -1.7, P > 0.05). The results of practical application showed that, the sensitivity and specificity of the qPCR were both 98.6%. The coincidence rate between the application results of the qPCR and the standard results was 98.6%, and their difference was not statistically significant (χ² = 0.0, P > 0.05). Conclusion The qPCR detection system using specific primers based on the 18S rDNA of Plasmodium spp. asexual stage has been established which can be applied in identifying malaria parasites in blood samples.

Key words: Plasomodium, Dye-qPCR, One-step qPCR, Detection threshold