PCR Fluorescent Probe-based Detection of Aspergillus Spp., Cryptococcus neoformans, and Pneumocystis jirovecii

Summary

Here, we present a clinical testing protocol based on the PCR fluorescent probe method for simultaneous detection of Aspergillus spp, Cryptococcus neoformans, and Pneumocystis jirovecii.

Abstract

This protocol describes a standardized procedure for the qualitative detection of Aspergillus spp., Cryptococcus neoformans, and Pneumocystis jirovecii in clinical sputum samples using a PCR fluorescent probe-based nucleic acid detection kit. The procedure involves clinical sample collection, alkaline liquefaction pretreatment, automated nucleic acid extraction, and multiplex real‑time PCR detection. Aspergillus spp., C. neoformans, and P. jirovecii are detected using target‑specific fluorescent probes (FAM, VIC, and CY5 channels, respectively), with an internal control (ROX channel) integrated for quality assurance. Assay validity and sample result interpretation are based on defined cycle threshold (Ct) cut-off values. A positive control must exhibit S-shaped amplification curves with Ct ≤ 33.7 in all channels, while the negative control must show no amplification. For clinical samples, a Ct value ≤ 33.7 in the FAM channel indicates positivity for Aspergillus spp., and a Ct value ≤ 36 in the VIC or CY5 channel indicates positivity for C. neoformans or P. jirovecii, respectively.

Introduction

Invasive fungal infections, such as those caused by Aspergillus spp., Cryptococcus neoformans, and Pneumocystis jirovecii, pose a significant threat to immunocompromised patients, leading to high morbidity and mortality. Timely and accurate diagnosis is critical for initiating appropriate antifungal therapy and improving clinical outcomes¹. Conventional diagnostic methods, including culture, microscopy, and antigen detection, have notable limitations. Culture is time-consuming and exhibits low sensitivity, microscopy lacks species-level identification and is operator-dependent, while antigen assays (e.g., galactomannan, β-D-glucan) may show cross-reactivity and inconsistent performance. Molecular diagnostics, particularly real-time PCR, can address some of these gaps by offering faster turnaround times and the potential for high sensitivity and specificity. Advances in multiplex PCR further allow simultaneous detection of multiple pathogens from a single sample, improving diagnostic efficiency.

This protocol describes a multiplex fluorescent probe PCR assay for qualitatively detecting DNA from Aspergillus spp., C. neoformans, and P. jirovecii in human sputum samples by targeting conserved regions of the 18S rRNA gene (Aspergillus spp.), the ITS gene (C. neoformans), and the mtLSU rRNA gene (P. jirovecii). Key features of this method include defined analytical sensitivity: The limit of detection for Aspergillus spp., C. neoformans, and P. jirovecii is 1,500 copies/mL; primer/probe specificity: target-specific primers and probes minimize cross-reactivity with human DNA or other common flora; robust contamination control: the incorporation of dUTP-UDG (uracil-DNA glycosylase) systems effectively prevents carryover contamination from previous PCR amplicons, a critical feature for maintaining assay reliability in a clinical laboratory setting²; integrated process control: an endogenous internal control targeting the human RP (Ribosomal Protein) gene, labeled with ROX fluorophore, is co-amplified to monitor nucleic acid extraction integrity and identify potential PCR inhibition, thereby reducing false-negative results³. This approach is supported by established guidelines for quality assurance in molecular diagnostics^4,5. The protocol is primarily applicable to respiratory specimens (sputum/BALF) from high-risk populations, such as hematology-oncology patients or transplant recipients. Potential limitations, including false positives from environmental contamination or reduced sensitivity due to excessive sample dilution, are also discussed, along with mitigation strategies.

Protocol

The use of clinical samples was approved by the appropriate ethics committee (Approval No.: ZYS-GCP-2025006). All participants provided broad informed consent for the use of their data and specimens in research. All reagents and consumables used in this protocol are listed in the Table of Materials.

1. Sample collection

1. Collect 3–5 mL of sputum or bronchoalveolar lavage fluid in sterile containers and submit for testing promptly.

2. Reagent preparation

NOTE: These steps were performed in the reagent preparation room.

1. Remove the detection kit from the -28 °C freezer and thaw completely at room temperature (25–30 °C) for 30 min.
2. Calculate the total number of reactions (N): N = number of test samples (n) + positive/negative controls (2) + 1
3. Prepare the reaction mix in a sterile microcentrifuge tube. For a single reaction: 35 μL of Nucleic Acid Amplification Mix + 5 μL of Primer-Probe Mix, vortex thoroughly, and briefly centrifuge.
4. Aliquot 40 μL of the reaction mix into each PCR tube (according to the kit manufacturer’s protocol) and transfer to the pass-through window of the sample processing room.

3. Sample processing

NOTE: These steps were performed in the Sample Processing Room. Waste Disposal: Discard supernatant from step 3.2.2 and other liquid waste containing NaOH into a designated chemical waste container.

1. Liquefaction
   1. Add sputum/bronchoalveolar lavage fluid (BALF) to a 50 mL screw-cap tube.
   2. Add 1–2 volumes of 4% NaOH digestion solution (relative to sputum viscosity).
      CAUTION: NaOH is corrosive. Avoid contact with skin and eyes.
   3. Tighten the cap, vortex for 1 min, and incubate in a biosafety cabinet at room temperature for 15–20 min.
2. Enrichment
   1. Centrifuge liquefied samples at 3,000 rpm for 5 min.
   2. Discard the supernatant using a pipette, retaining 1,000 μL of sediment.
   3. Vortex the sediment thoroughly.
3. Lysis
   1. Transfer 500 μL of sediment to a lysis tube. Vortex for 10 min and briefly centrifuge.
4. Extraction
   NOTE: Waste Disposal: Used extraction plates, tips, and tubes that contacted biological samples must be discarded into an approved biohazardous waste container.
   1. Transfer all lysate supernatant and 100 μL of negative/positive controls to Column 2 of the extraction plate.
   2. Perform nucleic acid extraction using an automated nucleic acid extraction system with the designated program⁶.
5. Sample loading
   1. Transfer 10 μL of extracted nucleic acid (Column 6) and controls to PCR tubes. Briefly centrifuge and transfer to the pass-through window of the amplification room.

4. Nucleic acid amplification

NOTE: These steps were performed in the Amplification Room.

1. Load the PCR tubes into the automated nucleic amplification instrument and select the appropriate amplification template.
2. Assign detection channels: Aspergillus spp.: FAM channel; C. neoformans: VIC channel; P. jirovecii: CY5 channel; Internal control: ROX channel^7,8,9.
3. Set sample names and run the program.

Results

Representative amplification curves obtained using this multiplex fluorescent probe PCR assay are shown in Figure 1 and Figure 2. Assay validity was determined based on predefined control criteria. In a valid run, the positive control produced characteristic S-shaped amplification curves in the FAM, VIC, CY5, and ROX channels, with cycle threshold (Ct) values ≤ 33.7 in all detection channels (Figure 1A). The negative control showed ...

Discussion

The multiplex real-time PCR protocol established in this study provides a standardized procedure for the simultaneous and rapid detection of three key invasive fungal pathogens (Aspergillus spp., Cryptococcus neoformans, and Pneumocystis jirovecii) in clinical sputum samples. This method enables the direct detection of pathogen DNA from clinical specimens. In contrast to traditional diagnostic approaches such as culture, microscopy, or serological antigen testing, this molecular method is not subject to the inherent limi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 μL Filter-Tip Sterile Extended Pipette Tips (Racked)	Xiaorui Biotechnology	S20221126
1000 μL Sterile Pipette Tips	Jiangsu Xinkang Medical Instrument Co.,Ltd	241101
200 μL Extended Filter Sterile Pipette Tips	Xiaorui Biotechnology	241001
Lysis Tubes	Beijing ZC Bio-Science & Technology Co	F1040
Nucleic Acid Detection Kit for Aspergillus spp, Cryptococcus neoformans, and Pneumocystis jirovecii	Beijing ZC Bio-Science & Technology Co.	CT8143-48T
Nucleic Acid Extraction Reagent	Beijing ZC Bio-Science & Technology Co	CN8053-B40T
Sample Dilution Buffer	Beijing ZC Bio-Science & Technology Co	SP7065