参见作者原研究论文

本实验方案简略版
Jun 2021
Advertisement

本文章节


 

A Rapid FRET Real-Time PCR Protocol for Simultaneous Quantitative Detection and Discrimination of Human Plasmodium Parasites
一种同时定量检测和鉴别人类疟原虫的快速FRET实时PCR方法   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Malaria is the most important parasitic disease worldwide, and accurate diagnosis and treatment without delay are essential for reducing morbidity and mortality, especially in P. falciparum malaria. Real-time PCR is highly sensitive and highly specific, therefore an excellent diagnostic tool for laboratory detection and species-specific diagnosis of malaria, especially in non-endemic regions where experience in microscopic malaria diagnostics is limited. In contrast to many other real-time PCR protocols, our new fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) allows the quantitative and species-specific detection of all human Plasmodium spp. in one run. Species identification is based on single nucleotide polymorphisms (SNPs) within the MalFL probe, detectable by melting curve analysis. A significant advantage of our FRET-qPCR is the short turn-around time of less than two hours, including DNA extraction, which qualifies it for routine diagnostics. Rapid and reliable species-specific malaria diagnosis is important, because treatment is species-dependent. Apart from first-line diagnosis, the quantitative results of our new FRET-qPCR can be helpful in therapy control, to detect early treatment failure.


Graphic abstract:



Keywords: Malaria (疟疾), Plasmodium (疟原虫), Diagnostics (诊断), PCR (PCR), Real-time PCR (实时荧光定量 PCR)

Background

Microscopy of Giemsa stained blood smears is still the gold standard for malaria diagnostics (Yin et al., 2018). However, microscopy requires considerable expertise, particularly at low-level parasitemia, which is often seen in imported malaria in non-endemic countries. Moreover, differentiating the Plasmodium species based on their morphological characteristics can be demanding, especially after chemoprophylaxis or auto-medication (Calderaro et al., 2018). Molecular detection and identification of Plasmodium spp. is a highly accurate and sensitive alternative method for the diagnosis of malaria. Particularly, real-time PCR has the advantage of fast and quantitative results and, compared to nested PCR, the risk of contamination is reduced. However, many published Plasmodium real-time PCR protocols have limitations, e.g., do not differentiate between species (Safeukui et al., 2008; Haanshuus et al., 2019; Farcas et al., 2004), do not detect all species (Perandin et al., 2004; Kim et al., 2014; Veron et al., 2009), or a positive genus-specific PCR has to be followed by two additional multiplex reactions, to obtain a species-specific result (Rougemont et al., 2004).


Hence, we developed and evaluated a new fluorescence resonance energy transfer-based real-time PCR (FRET-qPCR) that allows a quantitative, rapid, sensitive, and species-specific diagnosis of malaria (Schneider et al., 2021). The sequences of the hybridization probes MalFL and MalLC640 match P. falciparum. Fluorescence at 640 nm was generated by FRET, following the annealing of both probes to their adjacent complementary sequences. Species discrimination is based on the presence of single nucleotide polymorphisms (SNPs), reducing the affinity of the MalFLprobe for P. vivax/knowlesi (2 mismatches), P. ovale (1 mismatch), and P. malariae (1 mismatch), and thus lowering the melting temperature (Tm) in the melting curve analysis (Figure 1). The current paper is a step-by-step protocol for the performance of this new FRET-qPCR, focusing on the proper handling and storage of all components. The details described in this protocol are crucial comments to achieve excellent amplification and melting curves, a prerequisite for correct species identification based on single nucleotide polymorphisms (SNPs).


The advantage of requiring only one reaction per patient, and the short turn-around time of less than two hours (including DNA extraction), makes it a valuable diagnostic tool, especially in the absence of experienced microscopists. Moreover, results are objective and reproducible, DNA extraction can be automated, and the use of only one set of primers and probes is a significant advantage in routine clinical applications. One technician, well-trained in molecular methods, will be able to test a large number of patient samples in a relatively short turn-around time.


This protocol can be helpful for the rapid and reliable diagnosis of malaria in individual patients, particularly travelers returning from endemic areas, migrants, and refugees. Another field of application is treatment monitoring, by evaluating the quantitative results, and the crossing point (Cp) values. These allow the determination of the reduction in parasite number, important to detect early treatment failure (Rougemont et al., 2004). The potential of the FRET-qPCRs to detect asymptomatic infections can be useful for screening travelers returning to non-endemic regions. Moreover, it can also be performed in well-equipped malaria reference laboratories in endemic countries.

Materials and Reagents

  1. 1.5 mL microcentrifuge tubes, sterile (Sarstedt, catalog number: 72.690.01)

  2. 0.5–10 µL extra-long, 100–1,250 µL extra-long, 10–100 µL filtered pipette tips (Biozym, catalog numbers: VT0200, 770600; Eppendorf, catalog number: 30.077.547)

  3. QIAamp® DNA Mini Kit (Qiagen, catalog number: 51304)

  4. Ethanol absolute (Merck, catalog number: 1.00983.2500), do not use denatured alcohol

  5. DNase- and RNase free water (Promega, catalog number: P1193)

  6. LightCycler® Capillaries 20 µL (Roche, catalog number: 4929292001)

  7. LightCycler® Fast Start DNA Master HybProbe (Roche, catalog number: 12239272001)

  8. Primers and hybridization probes (TIB Molbiol GmbH; Table 1)

  9. Primer reconstitution and storage (working stock preparation instructions).

  10. EDTA blood samples


    Table 1. Primers and FRET-probes targeting a 157–165 bp fragment of the small subunit 18S rRNA gene, for the detection and simultaneous differentiation of the respective Plasmodium species.

    Oligonucleotide Sequence

    Plasmo 1a


    Plasmo 2a


    MalFLb



    MalLC640b


    5′-GTTAAGGGAGTGAAGACGATCAGA-3′


    5′-AACCCAAAGACTTTGATTTCTCATAA-3′


    5′-CTTTCATCCAACACCTAGTCGGC;

    3′label fluorescein


    5′-TAGTTTATGGTTAAGATTACGACGGT;

    5′label, LC red 640, 3′phosphorylated

    aPrimers Plasmo 1 and Plasmo 2 were published by Rougemont et al. (2004)

    bFRET-probes MalFL and MalLC640 were published by Schneider et al. (2021)

Equipment

  1. 0.5–10 µL, 10–100 µL, and 100–1,000 µL micropipettes (Eppendorf, catalog numbers: 3123000020, 3123000047, 3123000063)

  2. ThermoMixer (Eppendorf ThermoMixer C + Smart Block for 1.5 mL tubes; Eppendorf, catalog numbers: 5382000015, EPS360000036)

  3. Microcentrifuge (Thermo ScientificTM, PicoTM 21 Microcentrifuge, catalog number: 75002415)

  4. LightCycler® Instrument 2.0 (Roche, catalog number: 03531414001)

  5. LightCycler® Adapters (Roche, catalog number: 11909312001)

  6. Vortex (Vortex-Genie 2, Sigma-Aldrich, catalog number: Z258423)

Procedure

The preparation of primer stock solutions and the FRET-qPCR setup were carried out separately in different biosafety cabinets.

The DNA extraction should be performed on a clean working desk, that is only used for DNA extraction, with a separate set of pipettes, used only for this purpose. Additionally, false positive results are prevented by the use of aerosol-resistant pipette tips. During DNA extraction and FRET-qPCR setup, the gloves should be changed several times, to avoid cross contamination between different (sometimes high positive) samples. When removing the capillaries from the cycler after the PCR reaction is finished, take care that no capillary breaks! A broken capillary containing a positive sample bears the risk of a carry-over contamination, and has to be carefully cleaned, and gloves changed! Additionally, in the FastStart DNA Master HybProbe mix, dTTP is replaced by dUTP, allowing the use of uracil-N-glycosylase (UNG) as an additional carryover prevention measure.


  1. Human blood samples

    We recommend tubes containing ethylenediaminetetraacetic acid (EDTA) to prevent the coagulation of blood. In contrast to heparin (also used as anticoagulant), EDTA does not inhibit PCR and is frequently used for hematological, as well as molecular diagnostics. Gloves have to be worn all the time, because human blood samples might be infectious. Additionally, eye protection should be worn to minimise the chance of infection in case of aerosol generation, splashes, or other accidental spillages. Postal transport at room temperature should not exceed 48 h. Then, the blood samples should be stored at 4°C and DNA extraction should be performed the next day. Alternatively, store of the blood samples at -20°C until DNA extraction is possible.


  2. DNA extraction (QIAamp DNA Mini kit)

    Kit contents: QIAamp Mini Spin Columns, Collection tubes 2mL, Buffer AL, Buffer AW1 (concentrate), Buffer AW2 (concentrate), Buffer AE, Proteinase K. All components are stable for up to one year after delivery at room temperature (15–25°C).

    All centrifugation steps are performed at room temperature (15–25°C). Brief centrifugation means 5–10 s at maximum speed; this is sufficient to remove liquid from the lid, and collect the content at the bottom of the tube.

    1. Add absolute ethanol to Buffer AW1 and AW2, as indicated on the bottles.

    2. Pipet 20 µL of Proteinase K into the bottom of a 1.5-mL microcentrifuge tube.

    3. Add 200 µL of EDTA blood (invert the tube several times before pipetting the 200 µL!)

    4. Add 200 µL of Buffer AL, and mix by vortexing for 15 s.

      Note: Do not add Proteinase K directly to Buffer AL.

    5. Incubate in the ThermoMixer with shaking at 56°C for 10 min.

    6. Briefly centrifuge the tubes, to remove drops from the lid.

    7. Add 200 µL of absolute ethanol, vortex for 15 s, and briefly centrifuge the tubes.

    8. Carefully apply the mixture (620 µL) to the QIAamp Mini spin column (in a 2-mL collection tube). Close the cap and centrifuge at ≥6,000 × g for 1 min. Place the spin column in a fresh 2-mL collection tube, and discard the tube containing the filtrate.

    9. Add 500 µL of Buffer AW1, and centrifuge at ≥6,000 × g for 1 min.

    10. Discard the filtrate and the collection tube.

    11. Place the spin column in a fresh 2-mL collection tube, add 500 µL of Buffer AW2, and centrifuge at 14,000 × g for 3 min.

    12. Discard the filtrate and the collection tube.

    13. Place the spin column in a fresh 2-mL collection tube (not provided), and centrifuge at 14,000 × g for 1 min, to eliminate possible Buffer AW2 carryover.

    14. Place the spin column in a clean 1.5-mL microcentrifuge tube, add 100 µL of AE buffer, and incubate at room temperature for 5 min. Centrifuge at ≥6,000 × g for 1 min, to elute the purified DNA from the spin column.

    15. Store the DNA at 4°C until qPCR-setup (stable for several weeks at 4°C, or stable for 5 years when frozen at -20°C).


  3. FRET-qPCR

    1. Prepare primer and probe stock solutions (see working stock preparation instructions).

    2. The LightCycler® Fast Start DNA Master HybProbe kit is stored at -25–-15°C: Kit Contents: vial 1a (LightCycler® Fast Start Enzym), vial 1b (LightCycler® Fast Start reaction mix), vial 2 (MgCl2 stock solution, 25 mM), and vial 3 (H2O, PCR-grade).

    3. All centrifugation steps are at room temperature (15–25°C). Brief centrifugation means 5–10 s at maximum speed; this is sufficient to remove liquid from the lid, and collect the content at the bottom of the tube.

    4. Prepare the LightCycler® Fast Start DNA Master HybProbe (10× concentrated): thaw one vial 1a, and one vial 1b.

    5. Briefly centrifuge vial 1b and one vial 1a (enzyme).

    6. Pipet 60 µL from vial 1b into vial 1a, and mix gently by pipetting 5 times up and down. DO NOT vortex!

    7. Label vial 1a (red cap) with the new labels provided, it is now vial 1. Add the date; the reagent is stable at 4°C for 7 days.

    8. Thaw and mix vial 2 and vial 3, until all frozen parts have disappeared. Briefly centrifuge all tubes listed in Table 2.


      Table 2. FRET-qPCR mixture preparation

      Reagent Volume (µL) for one reaction

      Water, PCR-grade (vial 3)

      MgCl2 stock solution, 25 mM (vial 2)

      Plasmo 1 (20 µM)

      Plasmo 2 (20 µM)

      MalFL (4 µM)

      MalLC640 (4 µM)

      FastStart DNA Master HybProbe (vial 1)

      6.6

      2.4

      1.0

      1.0

      1.0

      1.0

      2.0

      Total volume 15.0


    9. Prepare the FRET-qPCR master mix for the required number of reactions plus one additional reaction, as described in Table 2.

    10. Mix all reagents carefully by pipetting up and down. DO NOT vortex the master mix!

    11. Briefly centrifuge the FRET-qPCR master mix.

    12. Take the precooled cooling block out of the fridge, and place the required number of capillaries into the precooled centrifuge adapters. Place the negative control at position 1 (PCR-grade water), and the positive control at position 2 (Plasmodium falciparum DNA), followed by the samples.

    13. Pipet 15 µL of mix into each precooled LightCycler capillary.

    14. Add 5 µL of DNA template, or respectively control.

    15. Seal each capillary with a stopper, immediately after adding the DNA (avoids mistakes).

    16. Place the adapters (containing the capillaries) into a standard benchtop microcentrifuge.

    17. Centrifuge at 700 × g (3,000 rpm) for exactly 8 s.

    18. Transfer the capillaries into the sample carousel of the LightCycler Instrument.

    19. Cycle the samples as described in Table 3.


      Table 3. LightCycler® 2.0 system FRET-qPCR Protocol

      Set all protocol parameters not listed in the table to “0”.

      Analysis Mode Cycles Target Temperature a Hold Time Acquisition Mode b
      None 1

      Denaturation

      95°C


      10 min


      none

      None 10

      Cycling (Touchdown)

      95°C

      69–58°C

      Slope =5°C/s

      72°C


      5 s

      10 s


      15 s


      none

      single


      none

      Quantification

      35

      Quantification

      95°C

      58°C

      Slope =5°C/s

      72°C


      5 s

      10 s


      15 s


      none

      single


      none

      Melting Curves 1

      Melting Curves

      95°C

      50°C

      70°C

      Slope =0.2°C/s


      20 s

      20 s

      0



      none

      none

      continuous


      None 1

      Cooling

      40°C


      30 s


      none


    aTemperature Transition Rate/Slope is 20°C/s, except where indicated.

    bDefault Channel 640 nm.

Data analysis

LightCycler® Software version 4.05 or higher

  1. Perform a Qualitative Detection Analysis, using controls.

  2. Capillary 1 is defined as a negative control and contains no DNA.

  3. Capillary 2 is defined as a positive control (Plasmodium falciparum DNA).

  4. If one of the controls fails to behave as expected, the unknown sample reactions are considered unreliable by the software: “Invalid”. If the controls perform as expected, the results for unknown samples are displayed as “Positive” or “Negative” in the combined column.

  5. The Cp value is automatically generated by the software for each positive sample, including the positive control.

  6. To perform a Melting Temperature analysis, select Tm calling from the Analysis toolbar. In the Melting Peaks display, the melting peaks and the calculated Tm values are displayed, and have to be controlled. Only when the software fails to calculate the Tm value properly, it shall be adjusted manually to peak maximum, using the manual Tm sliders in the Melting Peaks chart. (The difference in melting temperatures depends on the type of mismatch, the mismatch position within the probe sequence, and the base pairs immediately adjacent to the mismatch). According to the Tm value in the FRET-qPCR, the samples are assigned to P. falciparum (Tm 63.5–66°C), P. malariae (Tm 63.0–63.5°C), P. ovale (Tm 58–60°C), and P. vivax/knowlesi (Tm 56–57.5°C), respectively (Figure 1).

  7. Figure 1 shows the malaria species differentiation (P. vivax, P. ovale, P. malariae, and P. falciparum) based on the Tm values (Schneider et al., 2021).



    Figure 1. Species differentiation based on the Tm values.

    Melting curves of amplicons post real-time PCR from P. falciparum (red), P. malariae (green), P. ovale (lilac), and P. vivax (grey) (Schneider et al., 2021).


Working stock preparation instructions


Preparation of stock solution for primers and probes.

  1. The lyophilized oligonucleotides should be stored in the dark at room temperature or at 4°C. Do not freeze them before dissolving! They are stable at room temperature for at least one year.

  2. Briefly centrifuge the lyophilized primers Plasmo 1 and Plasmo 2 (exactly 5 nmol/tube), and probes MalFL and MalLC640 (exactly 1 nmol/tube), before opening in the safety cabinet.

  3. Add 250 µL of molecular-grade water to each tube.

  4. Vortex shortly and leave the tubes in the safety cabinet at room temperature for 30 min. Meanwhile, mix the tubes gently several times, and keep them away from direct sunlight.

  5. Briefly centrifuge and prepare aliquots, store them at -20°C.

  6. After thawing a set of aliquots, mix the four tubes gently, spin, and keep them away from excessive light. Store thawed primers and probes at 4°C, avoid multiple cycles of freezing and thawing. The shelf-life of the dissolved aliquots is at least 3 months at 4°C, or 1 year at -20°C.


Safety


When working with human blood samples, gloves and eye protection should be worn at all times to minimise the chance of infection.

Acknowledgments

This protocol is derived from our previous work (Schneider et al., 2021). The work was supported by the Medical University of Vienna, Austria.

Competing interests

There are no conflicts of interest or competing interests.

References

  1. Calderaro, A., Piccolo, G., Montecchini, S., Buttrini, M., Rossi, S., Dell'Anna, M. L., De Remigis, V., Arcangeletti, M. C., Chezzi, C. and De Conto, F. (2018). High prevalence of malaria in a non-endemic setting: comparison of diagnostic tools and patient outcome during a four-year survey (2013-2017). Malar J 17(1): 63.
  2. Farcas, G. A., Zhong, K. J., Mazzulli, T. and Kain, K. C. (2004). Evaluation of the RealArt Malaria LC real-time PCR assay for malaria diagnosis. J Clin Microbiol 42(2): 636-638.
  3. Haanshuus, C. G., Morch, K., Blomberg, B., Strom, G. E. A., Langeland, N., Hanevik, K. and Mohn, S. C. (2019). Assessment of malaria real-time PCR methods and application with focus on low-level parasitaemia. PLoS One 14(7): e0218982.
  4. Kim, J. Y., Goo, Y. K., Ji, S. Y., Shin, H. I., Han, E. T., Hong, Y., Chung, D. I., Cho, S. H. and Lee, W. J. (2014). Development and efficacy of real-time PCR in the diagnosis of vivax malaria using field samples in the Republic of Korea. PLoS One 9(8): e105871.
  5. Perandin, F., Manca, N., Calderaro, A., Piccolo, G., Galati, L., Ricci, L., Medici, M. C., Arcangeletti, M. C., Snounou, G., Dettori, G. and Chezzi, C. (2004). Development of a real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol 42(3): 1214-1219.
  6. Rougemont, M., Van Saanen, M., Sahli, R., Hinrikson, H. P., Bille, J. and Jaton, K. (2004). Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays. J Clin Microbiol 42(12): 5636-5643.
  7. Safeukui, I., Millet, P., Boucher, S., Melinard, L., Fregeville, F., Receveur, M. C., Pistone, T., Fialon, P., Vincendeau, P., Fleury, H., et al. (2008). Evaluation of FRET real-time PCR assay for rapid detection and differentiation of Plasmodium species in returning travellers and migrants. Malar J 7: 70.
  8. Schneider, R., Lamien-Meda, A., Auer, H., Wiedermann-Schmidt, U., Chiodini, P. L. and Walochnik, J. (2021). Validation of a novel FRET real-time PCR assay for simultaneous quantitative detection and discrimination of human Plasmodium parasites. PLoS One 16(6): e0252887.
  9. Veron, V., Simon, S. and Carme, B. (2009). Multiplex real-time PCR detection of P. falciparum, P. vivax and P. malariae in human blood samples. Exp Parasitol 121(4): 346-351.
  10. Yin, J., Li, M., Yan, H. and Zhou, S. (2018). Considerations on PCR-based methods for malaria diagnosis in China malaria diagnosis reference laboratory network. Biosci Trends 12(5): 510-514.

简介


[抽象的] 疟疾是全球最重要的寄生虫病,及时准确诊断和治疗对于降低发病率和死亡率至关重要,尤其是恶性疟。实时荧光定量 PCR 具有高灵敏度和高特异性,因此是疟疾实验室检测和物种特异性诊断的绝佳诊断工具,尤其是在显微疟疾诊断经验有限的非流行地区。与许多其他实时 PCR 协议相比,我们新的基于荧光共振能量转移的实时 PCR (FRET-qPCR) 允许对所有人类疟原虫进行定量和物种特异性检测。在一次运行。物种鉴定基于MalFL探针内的单核苷酸多态性 (SNP) ,可通过熔解曲线分析检测。我们的 FRET-qPCR 的一个显着优势是不到两个小时的周转时间,包括 DNA 提取,这使其有资格进行常规诊断。快速可靠的物种特异性疟疾诊断很重要,因为治疗取决于物种。除了一线诊断,我们新的 FRET-qPCR 的定量结果有助于治疗控制,检测早期治疗失败。

图文摘要:


[背景] 吉姆萨染色血涂片的显微镜检查仍然是疟疾诊断的金标准(Yin等人,2018 年)。然而,显微镜检查需要相当多的专业知识,特别是在低水平寄生虫血症方面,这在非流行国家的输入性疟疾中很常见。此外,根据形态特征区分疟原虫种类可能要求很高,尤其是在化学预防或自动用药后( Calderaro 等人,2018)。疟原虫的分子检测和鉴定。是一种高度准确和灵敏的疟疾诊断替代方法。特别是实时荧光定量 PCR 具有快速和定量结果的优势,与嵌套 PCR 相比,污染风险降低。然而,许多已发表的疟原虫实时 PCR 协议有局限性,例如,不区分物种( Safeukui 等人,2008;汉舒斯 等人,2019;法卡斯 et al ., 2004), 不检测所有物种 ( Perandin 等人,2004;金等人,2014;贝隆 et al ., 2009),或者必须在阳性的属特异性 PCR之后进行两个额外的多重反应,以获得物种特异性结果 (Rougemont et al ., 2004)。
因此,我们开发并评估了一种新的基于荧光共振能量转移的实时 PCR (FRET-qPCR),它可以对疟疾进行定量、快速、灵敏和物种特异性诊断(Schneider等人,2021 年)。 杂交探针MalFL和MalLC640的序列与恶性疟原虫匹配。在两个探针与它们相邻的互补序列退火后,FRET 产生了 640 nm 的荧光。物种区分基于单核苷酸多态性 (SNP) 的存在,从而降低MalFLprobe对间日疟原虫/诺氏疟原虫( 2 个错配) 、卵形疟原虫的亲和力 (1 个错配)和疟疾疟原虫(1 个错配),从而降低熔解曲线分析中的熔解温度 (Tm)(图 1)。目前的论文是这种新 FRET-qPCR 性能的分步协议,重点是所有组件的正确处理和存储。本协议中描述的细节是实现出色放大和熔解曲线的关键评论,这是基于单核苷酸多态性 (SNP) 进行正确物种鉴定的先决条件。
每位患者只需进行一次反应,以及不到两小时的周转时间(包括 DNA 提取),使其成为一种有价值的诊断工具,尤其是在没有经验丰富的显微镜检查人员的情况下。此外,结果客观且可重复,DNA提取可以自动化,并且仅使用一组引物和探针在常规临床应用中具有显着优势。一名在分子方法方面训练有素的技术人员将能够在相对较短的周转时间内测试大量患者样本。
该协议有助于快速和可靠地诊断个体患者的疟疾,特别是从流行地区返回的旅行者、移民和难民。另一个应用领域是治疗监测,通过评估定量结果和交叉点 (Cp) 值。这些允许确定寄生虫数量的减少,这对于检测早期治疗失败很重要(Rougemont等,2004)。 FRET-qPCR 检测无症状感染的潜力可用于筛查返回非流行地区的旅行者。此外,它还可以在流行国家设备齐全的疟疾参考实验室中进行。

关键字:疟疾, 疟原虫, 诊断, PCR, 实时荧光定量 PCR



材料和试剂


1.1.5 mL微量离心管,无菌( Sarstedt ,目录号:72.690.01)
2.0.5 – 10 µL 超长、100–1,250 µL 超长、10–100 µL 过滤移液器吸头( Biozym ,目录号:VT0200、770600;Eppendorf,目录号:30.077.547)
3.QIAamp ® DNA Mini Kit(Qiagen,目录号:51304)
4.无水乙醇(Merck,目录号:1.00983.2500),不要使用变性酒精
5.不含 DNase 和 RNase 的水(Promega,目录号:P1193)
6.LightCycler ®毛细管 20 µL(Roche,目录号:4929292001)
7.LightCycler ® Fast Start DNA Master HybProbe (Roche,目录号:12239272001)
8.引物和杂交探针(TIB Molbiol GmbH;表 1)
9.底漆重组和储存(工作库存准备说明)。
10.EDTA 血液样本


表 1. 针对小亚基 18S rRNA 基因的 157–165 bp 片段的引物和 FRET 探针,用于检测和同时区分相应的疟原虫物种。
寡核苷酸顺序
血浆1a _


血浆2a _


恶意软件b



MalLC640 b
5' - GTTAAGGGAGTGAAGACGATCAGA-3 '


5' - AACCCAAAGACTTTGATTTCTCATAA-3 '


5 ' -CTTTCATCCAACACCTAGTCGGC;
3 '标记荧光素


5 ' -TAGTTTATGGTTAAGATTACGACGGT;
5 '标记,LC red 640,3 '磷酸化
引物 Plasmo 1 和Plasmo 2 由 Rougemont等人出版。 (2004)
b FRET探针MalFL和 MalLC640 由 Schneider等人发表。 (2021)


设备


1.0.5–10 µL、10–100 µL 和 100–1,000 µL 微量移液器(Eppendorf,目录号:3123000020、3123000047、3123000063)
2.ThermoMixer (用于 1.5 mL 管的 Eppendorf ThermoMixer C + Smart Block;Eppendorf,目录号:5382000015,EPS360000036)
3.微量离心机(热 Scientific TM , Pico TM 21 微量离心机,目录号:75002415)
4.LightCycler ® Instrument 2.0(Roche,目录号:03531414001)
5.LightCycler ®适配器(Roche,目录号:11909312001)
6.Vortex(Vortex-Genie 2,Sigma-Aldrich,目录号:Z258423)


程序


引物储备溶液的制备和 FRET-qPCR 设置分别在不同的生物安全柜中进行。
DNA 提取应在干净的工作台上进行,该工作台仅用于 DNA 提取,并配有一套单独的移液器,仅用于此目的。此外,使用防气溶胶移液器吸头可防止假阳性结果。在 DNA 提取和 FRET-qPCR 设置期间,应多次更换手套,以避免不同(有时为高阳性)样品之间的交叉污染。在 PCR 反应完成后从循环仪中取出毛细管时,请注意不要损坏毛细管!含有阳性样品的破损毛细管有携带污染的风险,必须仔细清洁,并更换手套!此外,在 FastStart DNA Master HybProbe混合液中,dTTP 被dUTP取代,从而允许使用尿嘧啶-N-糖基化酶 (UNG) 作为额外的残留预防措施。


A.人体血液样本
我们推荐含有乙二胺四乙酸 (EDTA) 的试管以防止血液凝固。与肝素(也用作抗凝剂)相比,EDTA 不抑制 PCR,并且经常用于血液学和分子诊断。必须始终佩戴手套,因为人体血液样本可能具有传染性。此外,应佩戴护目镜,以尽量减少在气溶胶产生、飞溅或其他意外溢出时感染的机会。室温下的邮政运输不应超过 48 小时。然后,血样应储存在 4°C 下,第二天应进行 DNA 提取。或者,将血液样本储存在 -20°C 直到可以提取 DNA。


B.DNA 提取( QIAamp DNA Mini 试剂盒)
试剂盒内容: QIAamp Mini Spin Columns、2mL 收集管、Buffer AL、Buffer AW1(浓缩液)、Buffer AW2(浓缩液)、Buffer AE、Proteinase K。所有组分在室温下(15- 25℃)。
所有离心步骤均在室温(15–25°C) 下进行。短暂离心意味着以最大速度 5-10 秒;这足以从盖子中去除液体,并将内容物收集在管底部。
1.如瓶子上所示,将无水乙醇添加到缓冲液 AW1 和 AW2 中。
2.将 20 μL 的蛋白酶 K 移入 1.5 mL 微量离心管的底部。
3.添加 200 µL EDTA 血液(在移取 200 µL 之前将试管倒置几次!)
4.加入 200 μL 缓冲液 AL,涡旋15 秒混合。
注意:不要将蛋白酶 K 直接添加到 Buffer AL 中。
5.在ThermoMixer中孵育,在 56°C 下摇动 10 分钟。
6.短暂离心管子,从盖子上去除液滴。
7.加入 200 μL 的无水乙醇,涡旋 15 s,并短暂离心管。
8.小心地将混合物 (620 µL) 涂在QIAamp Mini 旋转柱上(在 2 mL 收集管中)。关闭盖子并在 ≥6,000 × g下离心1 分钟。将离心柱放入新的 2 mL 收集管中,并丢弃含有滤液的管。
9.加入 500 µL 缓冲液 AW1,并在 ≥6,000 × g下离心1 分钟。
10.丢弃滤液和收集管。
11.将离心柱放入新的 2 mL 收集管中,加入 500 µL Buffer AW2,以 14,000 × g离心3 分钟。
12.丢弃滤液和收集管。
13.将离心柱放入新的 2 mL 收集管(未提供)中,并以 14,000 × g离心1 分钟,以消除可能的 Buffer AW2 残留。
14.将旋转柱放入干净的 1.5 mL 微量离心管中,加入 100 μL AE 缓冲液,室温孵育5 分钟。以≥6,000 × g离心1 分钟,从离心柱中洗脱纯化的 DNA。
15.将 DNA 储存在 4°C 直到 qPCR 设置(在 4°C 下稳定数周,或在 -20°C 冷冻时稳定 5 年)。


C.FRET-qPCR
1.准备引物和探针原液(参见工作原液制备说明)。
2.LightCycler ® Fast Start DNA Master HybProbe试剂盒储存在 -25–-15°C: 试剂盒内容:小瓶 1a ( LightCycler ® Fast Start Enzym )、小瓶 1b ( LightCycler ® Fast Start 反应混合物)、小瓶 2 (MgCl 2 stock溶液,25 mM)和小瓶 3(H 2 O,PCR 级)。
3.所有离心步骤均在室温(15–25°C) 下进行。短暂离心意味着以最大速度 5-10 秒;这足以从盖子中去除液体,并将内容物收集在管底部。
4.准备LightCycler ® Fast Start DNA Master HybProbe (10 ×浓缩):解冻一个小瓶 1a 和一个小瓶 1b。
5.短暂离心小瓶 1b 和一个小瓶 1a(酶)。
6.从小瓶 1b 中吸取 60 µL 到小瓶 1a 中,上下吹打 5 次轻轻混匀。不要涡旋!
7.用提供的新标签标记小瓶 1a(红帽),现在是小瓶 1。添加日期;该试剂可在 4°C 下稳定 7 天。
8.解冻并混合小瓶 2 和小瓶 3,直到所有冷冻部分消失。简要离心表 2 中列出的所有管。


表 2. FRET-qPCR 混合物制备
试剂一次反应的体积 (µL)
水,PCR 级(小瓶 3)
MgCl 2储备溶液,25 mM(小瓶 2)
血浆1 (20 µM)
等离子2 (20 µM)
MalFL (4 µM)
MalLC640 (4 µM)
FastStart DNA Master HybProbe (小瓶 1)6.6
2.4
1.0
1.0
1.0
1.0
2.0
总容积15.0


9.如表 2 中所述,为所需的反应数量加上一个额外的反应准备 FRET-qPCR 主混合物。
10.通过上下移液小心混合所有试剂。不要涡旋混合主混合物!
11.短暂离心 FRET-qPCR 主混合物。
12.将预冷的冷却块从冰箱中取出,并将所需数量的毛细管放入预冷的离心机适配器中。将阴性对照置于位置 1(PCR 级水),将阳性对照置于位置 2(恶性疟原虫DNA),然后是样品。
13.吸取 15 μL 的混合物到每个预冷的LightCycler毛细管中。
14.添加 5 μL 的 DNA 模板,或分别控制。
15.添加 DNA 后立即用塞子密封每个毛细管(避免错误)。
16.将适配器(包含毛细管)放入标准台式微量离心机中。
17.以 700 × g (3,000 rpm) 的速度离心8 秒。
18.将毛细管转移到LightCycler Instrument 的样品转盘中。
19.如表 3 中所述循环样品。


表 3. LightCycler ® 2.0 系统 FRET-qPCR 协议
将表中未列出的所有协议参数设置为“0”。
分析模式循环目标温度a保持时间采集模式b
没有1变性
95°C
10 分钟
没有
没有10骑自行车(触地得分)
95°C
69–58°C
斜率 =5°C/秒
72°C
5 秒
10 秒


15 秒
没有
单身的


没有


量化
35量化
95°C
58°C
斜率 =5°C/秒
72°C
5 秒
10 秒


15 秒
没有
单身的


没有
熔化曲线1熔化曲线
95°C
50°C
70°C
斜率 =0.2°C/秒20 秒
20 秒
0

没有
没有
连续


没有1冷却
40°C
30 秒
没有
a温度转变速率/斜率是 20°C/秒,除非另有说明。
b默认通道 640 nm。


数据分析


LightCycler ®软件版本 4.05 或更高版本
1.使用对照进行定性检测分析。
2.毛细管 1 被定义为阴性对照,不含 DNA 。 
3.毛细管 2 定义为阳性对照(恶性疟原虫DNA)。
4.如果其中一个控制未能按预期运行,则软件认为未知的样品反应不可靠:“无效”。如果控制按预期执行,未知样品的结果将在组合列中显示为“阳性”或“阴性”。
5.软件会自动为每个阳性样本(包括阳性对照)生成 Cp 值。
6.要执行熔化温度分析,请从分析工具栏中选择 Tm 调用。在熔解峰显示中,熔解峰和计算的 Tm 值会显示出来,必须加以控制。只有当软件无法正确计算 Tm 值时,才应使用熔化峰图表中的手动 Tm 滑块手动调整到峰值最大值。 (解链温度的差异取决于错配的类型、探针序列内的错配位置以及与错配直接相邻的碱基对)。根据 FRET-qPCR 中的 Tm 值,将样本分配给恶性疟原虫(Tm 63.5–66°C)、疟疾疟原虫( Tm 63.0–63.5°C) 、卵形疟原虫(Tm 58–60°C ) ) 和间日疟原虫/诺氏疟原虫(Tm 56–57.5°C) 分别(图 1)。
7.基于 Tm 值的疟疾物种分化(间日疟原虫、卵形疟原虫、疟原虫和恶性疟原虫) ( Schneider等人,2021)。


 
图 1. 基于 Tm 值的物种分化。 
恶性疟原虫(红色)、疟疾疟原虫(绿色)、卵形疟原虫(淡紫色)和间日疟原虫(灰色)的实时 PCR 后扩增子的熔解曲线(Schneider等人,2021)。


工作库存准备说明
为引物和探针制备储备溶液。
1.冻干的寡核苷酸应在室温或 4°C 下避光储存。溶解前不要冷冻它们!它们在室温下稳定至少一年。
2.在安全柜中打开之前,短暂离心冻干引物Plasmo 1 和Plasmo 2(精确到 5 nmol/管)和探针MalFL和 MalLC640(精确到 1 nmol/管)。
3.在每管中加入 250 μL 的分子级水。
4.短暂涡旋,将管子在室温下放置在安全柜中 30 分钟。同时,轻轻混合试管数次,避免阳光直射。
5.短暂离心并制备等分试样,将它们储存在-20°C。
6.解冻一组等分试样后,轻轻混合四管,旋转,并使其远离过度光照。将解冻的引物和探针储存在 4°C,避免多次冻融循环。溶解的等分试样的保质期在 4°C 下至少为 3 个月,在 -20°C 下至少为 1 年。


安全
佩戴手套和护目镜,以尽量减少感染机会。


致谢


该协议源自我们之前的工作 (Schneider et al ., 2021)。这项工作得到了奥地利维也纳医科大学的支持。


利益争夺


不存在利益冲突或竞争利益。


参考


1.Calderaro , A., Piccolo, G., Montecchini , S., Buttrini , M., Rossi, S., Dell'Anna , ML, De Remigis , V., Arcangeletti , MC, Chezzi , C. 和 De Conto , F (2018 年)。非流行环境中疟疾的高患病率:四年调查(2013-2017 年)期间诊断工具和患者结果的比较。 颧骨 J 17(1):63。
2.Farcas , GA, Zhong, KJ, Mazzulli , T. 和 Kain, KC (2004)。评估用于疟疾诊断的 RealArt Malaria LC 实时 PCR 测定。 临床微生物杂志 42(2):636-638。
3.Haanshuus , CG, Morch , K., Blomberg, B., Strom, GEA, Langeland , N., Hanevik , K. 和Mohn , SC (2019)。评估疟疾实时 PCR 方法和应用,重点关注低水平寄生虫血症。 公共科学图书馆一号14(7):e0218982。
4.Kim, JY, Goo, YK, Ji, SY, Shin, HI, Han, ET, Hong, Y., Chung, DI, Cho, SH 和 Lee, WJ (2014)。在大韩民国使用现场样本诊断间日疟的实时 PCR 技术的发展和有效性。 公共科学图书馆一号9(8):e105871。
5.Perandin , F., Manca , N., Calderaro , A., Piccolo, G., Galati, L., Ricci, L., Medici, MC, Arcangeletti , MC, Snounou , G., Dettori , G. and Chezzi , C.(2004 年)。开发用于检测恶性疟原虫、间日疟原虫和卵形疟原虫的实时 PCR 检测方法,用于常规临床诊断。 临床微生物杂志 42(3):1214-1219。
6.Rougemont, M., Van Saanen, M., Sahli , R., Hinrikson , HP, Bille , J. 和Jaton , K. (2004)。通过基于 18S rRNA 基因亚基和物种特异性实时 PCR 测定法检测人类血液中的四种疟原虫。 临床微生物杂志 42(12):5636-5643。
7.Safeukui , I., Millet, P., Boucher, S., Melinard , L., Fregeville , F., Receveur , MC, Pistone , T., Fialon , P., Vincendeau , P., Fleury, H.等人_ (2008 年)。评估 FRET 实时 PCR 检测快速检测和区分返回的旅行者和移民中的疟原虫种类。 颧骨 J 7:70 。
8.Schneider, R.、Lamien-Meda, A.、Auer, H.、Wiedermann-Schmidt, U.、Chiodini, PL 和 Walochnik, J. (2021)。验证一种用于同时定量检测和鉴别人类疟原虫寄生虫的新型 FRET 实时 PCR 测定。 公共科学图书馆一号16(6):e0252887。
9.Veron , V.、Simon, S. 和 Carme, B. (2009)。人类血液样本中恶性疟原虫、间日疟原虫和疟疾疟原虫的多重实时 PCR 检测。 Exp Parasitol 121(4):346-351。
10.Yin, J.、Li, M.、Yan, H. 和 Zhou, S.(2018 年)。中国疟疾诊断参考实验室网络基于PCR的疟疾诊断方法思考[J]. Biosci趋势12(5):510-514。


登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用:Schneider, R., Lamien-Meda, A., Auer, H., Wiedermann-Schmidt, U., Chiodini, P. L. and Walochnik, J. (2022). A Rapid FRET Real-Time PCR Protocol for Simultaneous Quantitative Detection and Discrimination of Human Plasmodium Parasites. Bio-protocol 12(7): e4381. DOI: 10.21769/BioProtoc.4381.
分类
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。