Fungi are eukaryotic microorganisms whose cell walls contain chitin and β‑glucans. The fungal cell wall component 1,3‑β‑D‑glucan can specifically activate Factor G in the limulus amebocyte lysate, triggering a cascade of enzymatic reactions that ultimately react with a chromogenic substrate—limulus tripeptide‑PNA conjugate—yielding a measurable optical signal at a specific wavelength. This enables the quantitative detection of 1,3‑β‑D‑glucan, a method also known as the G test (colorimetric assay). The G test is capable of detecting infections caused by various fungi, including Candida, Aspergillus, Pneumocystis, Fusarium, Geotrichum, and Trichosporon, among others. The following are key operational considerations for performing the G test:

Pre-analytical quality assurance:

1. Collect serum strictly in accordance with the operating procedures: perform aseptic techniques, using dedicated pyrogen‑free vacuum blood collection tubes (BD red‑top tubes containing a clotting activator), and ensure that all instruments, including the tubes, are sterile and free of pyrogens.

2. Within 2 hours after blood collection, separate the serum by centrifuging at 3,000 rpm (1,600 × g) for 15 minutes. If immediate analysis is not possible, transfer the serum to a pyrogen‑free transfer tube and store it frozen at −20°C; use within one week.

3. For routine patients, draw blood in the morning before medication administration (for hemodialysis patients, draw blood prior to dialysis).

Quality Assurance in Analysis:

1. Perform procedures in a clean environment, use powder-free latex gloves, wear a mask, and don a lab coat.

2. Use disposable, pyrogen‑free pipette tips; ensure the tips do not come into contact with contaminants, prevent glass fragments from falling into the ampoules, and avoid touching the mouth of the glass vials with your hands.

3. During sampling, the middle-upper layer of the serum should be collected, and after sample processing, the middle-upper layer should also be retained.

4. Avoid strong air currents, and perform intubation gently; vibrations may affect the reaction curve.

5. Jaundice, hemolysis, and chylous blood should be considered unacceptable specimens; if clinically indicated, they may be tested after being diluted 2–5 fold with pyrogen‑free assay water or sample‑processing solution manufactured by a specialized company.

The G test is an important microbiological criterion in fungal diagnostic standards and serves as a clinical indicator for invasive fungal infections. By carefully observing the necessary precautions during testing, the accuracy and speed of G‑test results can be ensured.