Establishment and application of a specific primers-based qPCR targeting asexual stage 18S rDNA for detecting five Plasmodium species

doi:10.12140/j.issn.1000-7423.2023.01.006

Abstract

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

CLC Number:

R382.31

LI Mei, XIAO Ning, XIA Zhigui. Establishment and application of a specific primers-based qPCR targeting asexual stage 18S rDNA for detecting five Plasmodium species[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2023, 41(1): 36-43.

Figures/Tables 6

Fig. 1

Table 1

Table 2

Table 3

Table 4

Table 5

References 25

[1]	Zhang L, Feng J, Zhang SS, et al. The progress of national malaria elimination and epidemiological characteristics of malaria in China in 2017[J]. Chin J Parasitol Parasit Dis, 2018, 36(3): 201-209. (in Chinese)
	(张丽, 丰俊, 张少森, 等. 2017年全国消除疟疾进展及疫情特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(3): 201-209.)
[2]	Zhang L, Feng J, Zhang SS, et al. Epidemiological characteristics of malaria and the progress towards its elimination in China in 2018[J]. Chin J Parasitol Parasit Dis, 2019, 37(3): 241-247. (in Chinese)
	(张丽, 丰俊, 张少森, 等. 2018年全国疟疾疫情特征及消除工作进展[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(3): 241-247.)
[3]	Zhang L, Feng J, Xia ZG, et al. Epidemiological characteristics of malaria and progress on its elimination in China in 2019[J]. Chin J Parasitol Parasit Dis, 2020, 38(2): 133-138. (in Chinese)
	(张丽, 丰俊, 夏志贵, 等. 2019年全国疟疾疫情特征分析及消除工作进展[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 133-138.)
[4]	Zhang L, Feng J, Tu H, et al. Malaria epidemiology in China in 2020[J]. Chin J Parasitol Parasit Dis, 2021, 39(2): 195-199. (in Chinese)
	(张丽, 丰俊, 涂宏, 等. 2020年全国疟疾疫情分析[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(2): 195-199.)
[5]	Zhang L, Yi BY, Xia ZG, et al. Epidemiological characteristics of malaria in China, 2021[J]. Chin J Parasitol Parasit Dis, 2022, 40(2): 135-139. (in Chinese)
	(张丽, 易博禹, 夏志贵, 等. 2021年全国疟疾疫情特征分析[J]. 中国寄生虫学与寄生虫病杂志, 2022, 40(2): 135-139.)
[6]	World Health Organization. World malaria report[R]. Geneva: WHO, 2022.
[7]	Huang LY. Principle, method and application of the latest PCR technology[M]. Beijing: Chemical Industry Press, 2005:52-67. (in Chinese)
	(黄留玉. PCR最新技术原理、方法及应用[M]. 北京: 化学工业出版社, 2005: 52-67.)
[8]	Yuan YN, Liu WZ. The types, characteristics and applications of real-time fluoescent quantitative PCR techniques[J]. Chin Animal Husb ＆ Vet Med, 2008, 35(3): 27-30. (in Chinese)
	(袁亚男, 刘文忠. 实时荧光定量PCR技术的类型、特点与应用[J]. 中国畜牧兽医, 2008, 35(3): 27-30.)
[9]	Haanshuus C, Mohn S. Plasmodium genus- and species-specific real-time PCR using SYBR dye decreases laboratory time without impairing the sensitivity or specificity compared to conventional PCR[J]. Malar J, 2014, 13(suppl 1): 39. doi: 10.1186/1475-2875-13-39
[10]	Abdullah NR, Furuta T, Taib R, et al. Short report: development of a new diagnostic method for Plasmodium falciparum infection using a reverse transcriptase-polymerase chain reaction[J]. Am J Trop Med Hyg, 1996, 54(2): 162-163. pmid: 8619441
[11]	Kamau E, Tolbert LS, Kortepeter L, et al. Development of a highly sensitive genus-specific quantitative reverse transcriptase real-time PCR assay for detection and quantitation of Plasmodium by amplifying RNA and DNA of the 18S rRNA genes[J]. J Clin Microbiol, 2011, 49(8): 2946-2953. doi: 10.1128/JCM.00276-11
[12]	Hodgson SH, Douglas AD, Edwards NJ, et al. Increased sample volume and use of quantitative reverse-transcription PCR can improve prediction of liver-to-blood inoculum size in controlled human malaria infection studies[J]. Malar J, 2015, 14: 33. doi: 10.1186/s12936-015-0541-6
[13]	Kefi M, Mavridis K, Simões ML, et al. New rapid one-step PCR diagnostic assay for Plasmodium falciparum infective mosquitoes[J]. Sci Rep, 2018, 8: 1462. doi: 10.1038/s41598-018-19780-6
[14]	Çavuş İ, Özbilgin A, Balcıoğlu İC. The follow-up of treatment process of malaria by real-time polymerase chain reaction: in vivo model[J]. Mikrobiyol Bul, 2021, 55(4): 603-616. doi: 10.5578/mb.20219711
[15]	Li J, Wirtz RA, McConkey GA, et al. Plasmodium: genus-conserved primers for species identification and quantitation[J]. Exp Parasitol, 1995, 81(2): 182-190. pmid: 7556560
[16]	Snounou G, Viriyakosol S, Jarra W, et al. Identification of the four human malaria parasite species in field samples by the polymerase chain reaction and detection of a high prevalence of mixed infections[J]. Mol Biochem Parasitol, 1993, 58(2): 283-292. doi: 10.1016/0166-6851(93)90050-8
[17]	Li M, Wang ZY, Zhang T, et al. Exploration of using one-step reverse transcription PCR in detection of four species of human malaria parasites[J]. Chin J Parasitol Parasit Dis, 2016, 34(6): 500-505. (in Chinese)
	(李美, 王真瑜, 张淘, 等. 一步反转录PCR技术在检测4种人疟原虫中的初步应用[J]. 中国寄生虫学与寄生虫病杂志, 2016, 34(6): 500-505.)
[18]	Chen JT, Liu PF, Zhong DS, et al. Quantitative detection and species identification of human Plasmodium spp. by SYBR Green Ⅰ real-time PCR[J]. J Mol Diagn Ther, 2013, 5(4): 227-231. (in Chinese)
	(陈江涛, 刘配芬, 钟德善, 等. SYBR Green Ⅰ染料法定量PCR检测疟原虫感染及[J]. 分子诊断与治疗杂志, 2013, 5(4): 227-231.)
[19]	Mangold KA, Manson RU, Koay ESC, et al. Real-time PCR for detection and identification of Plasmodium spp.[J]. J Clin Microbiol, 2005, 43(5): 2435-2440. doi: 10.1128/JCM.43.5.2435-2440.2005 pmid: 15872277
[20]	Wang SQ, Zhou HY, Li Z, et al. Quantitative detection and species identificaton of human Plasmodium spp. by using SYBR Green Ⅰ based real-time PCR[J]. Chin J Schisto Control, 2011, 23(6): 677-681. (in Chinese)
	(汪圣强, 周华云, 李宗, 等. SYBR Green Ⅰ染料法定量PCR用于人体疟原虫定量检测及虫种鉴别的研究[J]. 中国血吸虫病防治杂志, 2011, 23(6): 677-681.)
[21]	Oddoux O, Debourgogne A, Kantele A, et al. Identification of the five human Plasmodium species including P. knowlesi by real-time polymerase chain reaction[J]. Eur J Clin Microbiol Infect Dis, 2011, 30(4): 597-601. doi: 10.1007/s10096-010-1126-5
[22]	Li H. SYBR green real time PCR assay for rapid detection of Plasmodium falciparum and Plasmodium vivax[D]. Changsha: Central South University, 2014: 14-19. (in Chinese)
	(李欢. SYBR Green实时PCR检测恶性疟原虫与间日疟原虫方法的建立[D]. 长沙: 中南大学, 2014: 14-19.)
[23]	Li M, Xia ZG, Zhou SS. Analysis of inconsistence of Plasmodium detection in some malaria cases[J]. Chin J Parasitol Parasit Dis, 2019, 37(4): 464-471. (in Chinese)
	(李美, 夏志贵, 周水森. 疟疾病例复核确认过程中检测结果不一致常见问题分析[J]. 中国寄生虫学与寄生虫病杂志, 2019, 37(4): 464-471.)
[24]	Taylor BJ, Lanke K, Banman SL, et al. A direct from blood reverse transcriptase polymerase chain reaction assay for monitoring falciparum malaria parasite transmission in elimination settings[J]. Am J Trop Med Hyg, 2017, 97(2): 533-543. doi: 10.4269/ajtmh.17-0039
[25]	Haanshuus CG, Mørch K, Blomberg B, et al. Assessment of malaria real-time PCR methods and application with focus on low-level parasitaemia[J]. PLoS One, 2019, 14(7): e0218982. doi: 10.1371/journal.pone.0218982

保守区编号 Code of conservative area	在Pf-A上的位置 Location on Pf-A	片段长度/bp Fragment length/bp	一致性/% Identification/%
保守区编号 Code of conservative area	在Pf-A上的位置 Location on Pf-A	片段长度/bp Fragment length/bp	Po-S	Pf-S	其他10条 Other 10 sequences
C1	1~65	65	95	98	100
C2	85~126	42	98	98	98
C3	298~480	183	96	98	≥ 99
C4	550~644	95	99	98	100
C5	942~1 100	159	93	94	≥ 97
C6	1 184~1 454	271	98	99	100
C7	1 638~1 724	87	92	97	100
C8	1 831~1 978	148	92	97	≥ 98
C9	2 003~2 092	90	92	97	100

引物名称 Primer name		引物序列（5´→3´） Primer sequence (5´→3´)	在18S rDNA上的位置 Location on 18S rDNA	片段长度/bp Fragment length/bp	Tm值^a/℃ Tm value^a /℃	扩增效率/% Amplification efficiency/%
疟原虫属 Plasmodium spp.	Pg-F	GCGCAAGCGAGAAAGTTAAAAG	C6	145	82	98.8
疟原虫属 Plasmodium spp.	Pg-R	CCGCTAATTAGCAGGTTAAGAT	C6	145	82	98.8
Pf	Pf-F	TTATTATCCTTTGATTTTTATCTTTGG	V2	108	72	90.6
Pf	Pf-R	ATAAATTTATTACGTGTTACTTCTTTG	V2	108	72	90.6
Pv^[17]	Pv-F	TTTTAAGGACTTTCTTTGCTTCGG	V3	321	79.5 ~ 80.0	92.7
Pv^[17]	Pv-R	CGACGGTATCTGATCGTCTTC	C4	321	79.5 ~ 80.0	92.7
Pm	Pm-F	CAAGCGTTATATTTTTTCTGTTACAT	V3	165	73.5 ~ 74.5	96.0
Pm	Pm-R	GAAACTGTTTTAACTGTTTGCTTTG	V3	165	73.5 ~ 74.5	96.0
Po	Po-F	TATAAGATGCTTAGGCAATACAAC	V3	177	76.0 ~ 77.5	97.3
Po	Po-R	TCAAACGCAGTAAAATGCTTTAACT	V3	177	76.0 ~ 77.5	97.3
Pk	Pk-F	GAAGAAAATATTGGAATTACGTTAAAT	V5	90	73.5 ~ 74.0	95.7
Pk	Pk-R	CATTCATACGCAAAAAAGAAATAGAT	V5	90	73.5 ~ 74.0	95.7

引物对 Primer pairs	非目标DNA的最高浓度/拷贝·μl^-1The highest concentration of non-target DNA/copy·μl^-1
引物对 Primer pairs	Pf	Pv	Pm	Po	Pk
Pf-F/Pf-R		5.0 × 10⁸	5.0 × 10⁷	5.0 × 10⁷	5.0 × 10⁷
Pv-F/Pv-R	5.0 × 10⁸		5.0 × 10⁸	5.0 × 10⁸	5.0 × 10⁷
Pm-F/Pm-R	5.0 × 10⁶	5.0 × 10⁶		5.0 × 10⁶	5.0 × 10⁷
Po-F/Po-R	5.0 × 10⁷	5.0 × 10⁶	5.0 × 10⁶		5.0 × 10⁷
Pk-F/Pk-R	5.0 × 10⁶	5.0 × 10⁶	5.0 × 10⁷	5.0 × 10⁷

引物对 Primer pairs	目标DNA Target DNA				目标和非目标DNA混合 Mix of target DNA and non-target DNA
	5.0 × 10²拷贝·μl^-1 5.0 × 10²copy·μl^-1		5.0 × 10⁶拷贝·μl^-1 5.0 × 10⁶copy·μl^-1		5.0 × 10²拷贝·μl^-1 5.0 × 10²copy·μl^-1
	平均Ct值 Average Ct values	变异系数/% Coefficient of variation/%	平均Ct值 Average Ct values	变异系数/% Coefficient of variation/%	非目标DNA浓度/拷贝·μl^-1 Concentration of non-target DNA/copy·μl^-1	平均Ct值 Average Ct values	变异系数/% Coefficient of variation/%
Pf-F/Pf-R	34.1 ± 0.8	2.5	19.9 ± 0.0	0.1	5.0 × 10⁶	34.3 ± 0.1	0.3
Pv-F/Pv-R	30.6 ± 0.5	1.5	17.5 ± 0.0	0.0	5.0 × 10⁷	31.6 ± 0.9	3.0
Pm-F/Pm-R	32.1 ± 0.0	0.1	18.4 ± 0.1	0.4	5.0 × 10⁶	32.6 ± 0.4	1.4
Po-F/Po-R	31.7 ± 0.7	2.1	17.6 ± 0.0	0.0	5.0 × 10⁷	31.4 ± 0.4	1.2
Pk-F/Pk-R	33.6 ± 0.1	0.3	19.4 ± 0.0	0.0	5.0 × 10⁶	33.4 ± 0.4	1.2

引物对 Primer pairs	样品 Sample	最低检出原虫密度/虫·μl^-1 Minimum parasite density/Parasite·μl^-1
引物对 Primer pairs	样品 Sample	一步反转录qPCR One-step reverse transcription qPCR	qPCR	巢式PCR Nest PCR
Pg-F/Pg-R	X差值^a最大的恶性疟原虫样品 P. falciparum sample with maximum D-value of X^a	0.08	2.00
Pf-F/Pf-R	X差值最大的恶性疟原虫样品 P. falciparum sample with maximum D-value of X	0.02	2.00	9.9
Pf-F/Pf-R	X差值最小的恶性疟原虫样品 P. falciparum sample with mimimum D-value of X	0.40	0.40	2.0
Pv-F/Pv-R	X差值最大的间日疟原虫样品 P. vivax sample with maximum D-value of X	0.01	0.30	6.9
Pv-F/Pv-R	X差值最小的间日疟原虫样品 P. vivax sample with mimimum D-value of X	10.80	10.80	10.8
Pm-F/Pm-R	X差值最大的三日疟原虫样品 P. malariae sample with maximum D-value of X	0.30	7.40	7.4
Pm-F/Pm-R	X差值最小的三日疟原虫样品 P. malariae sample with mimimum D-value of X	10.6	10.60	10.6
Po-F/Po-R	X差值最大的卵形疟原虫样品 P. ovale sample with maximum D-value of X	0.06	7.70	7.7
Po-F/Po-R	X差值最小的卵形疟原虫样品 P. ovale sample with mimimum D-value of X	10.00	10.00	10.0
Pk-F/Pk-R	诺氏疟原虫样品 P. knowlesi sample	8.30	8.30
	平均最低检出限 Everage detection limit	4.1 ± 5.1	6.0 ± 4.3	8.2 ± 2.9