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., galactomanna....

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 r....

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