iFIND Technology Analysis

Molecular Beacon Melting Curve Analysis: A Breakthrough for Rifampicin Resistance Detection in Tuberculosis

1. Severe Impact of Drug-Resistant Tuberculosis

According to the Global Tuberculosis Report 2024, China had an estimated 748,000 new tuberculosis cases in 2023 (incidence rate: 52 per 100,000 population), including 29,000 cases of rifampicin-resistant tuberculosis (incidence rate: 2.1 per 100,000). Although the etiological positivity rate in China has reached 67%, and rifampicin resistance screening rates for new and retreated patients have increased to 89% and 91% respectively, only 13,400 resistant cases were diagnosed and reported based on WHO estimates, indicating a continued need to strengthen detection capacity for drug-resistant tuberculosis.

The National Tuberculosis Prevention and Control Plan (2024–2030) sets a goal to increase the drug resistance testing rate among etiologically positive patients to 95% by 2030 to support precise diagnosis and standardized treatment management. Meanwhile, the Chinese Center for Disease Control and Prevention requires that all county/district designated medical institutions be equipped with user-friendly molecular biological drug resistance testing devices within 3 years to comprehensively improve primary-level testing capacity.

2. Challenges in Tuberculosis Drug Resistance Detection

Traditional phenotypic drug susceptibility testing based on mycobacterial culture provides comprehensive and reliable results but requires culture-positive isolates and is time-consuming (6–8 weeks for solid culture, approximately 20 days for liquid culture). It also demands strict laboratory conditions and biosafety levels, making it unsuitable for initial screening of drug resistance.

In contrast, molecular detection offers high sensitivity and specificity, with results available within 3 hours, making it ideal for rapid initial screening of tuberculosis drug resistance.

The primary molecular target for rifampicin resistance detection is the rpoB gene of Mycobacterium tuberculosis, especially the 81-bp core mutation region (DDRD) where most rifampicin resistance-associated mutations occur. However, this region contains numerous mutation sites, and conventional detection methods carry a risk of missed detection. A fully covering detection technology is therefore essential to ensure all resistance mutations are reliably identified.

(Selected rifampicin resistance mutations listed by WHO)

3. Advantages of the Molecular Beacon Melting Curve Method

The iFIND TBR technology uses 4 specific molecular beacon probes that fully cover the 81-bp core region of the rpoB gene, enabling mutation detection through high-precision melting curve analysis.

The principle is as follows: double-stranded DNA is stabilized by hydrogen bonds and base stacking forces. During heating, hydrogen bonds break and strands dissociate. At the temperature where 50% of duplexes are dissociated, the fluorescent signal changes abruptly; this temperature is defined as the Tm value. Tm depends on intrinsic properties such as GC content and probe length, and remains stable when the probe and target sequence are perfectly matched.

If a mutation occurs in the target, binding between the probe and target is weakened, causing a shift in Tm, allowing precise detection of the mutation. This method demands extremely high thermal control precision. The iFIND system delivers excellent temperature control with a module precision ≤0.1℃ and Tm resolution up to 0.01℃/s.

Since molecular beacon melting curves are more complex than traditional dye-based methods and Tm interpretation is less intuitive, Rocgene has developed a dedicated intelligent analysis algorithm that accurately identifies Tm shifts. Theoretically, this technology can detect all mutation types, including single-point mutations, double-point mutations, and deletions, providing a highly efficient and reliable solution for Mycobacterium tuberculosis drug resistance detection.

(Test results of 143 strains with various mutations and wild type)

In addition, this method offers unique advantages in detecting heteroresistance. When both resistant and susceptible strains are present in a sample, the melting curve shows a Tm shift or double peaks, allowing effective identification of low-proportion resistant strains and preventing missed detection.

(Double peaks indicating heteroresistance)

In the future, with big data accumulation and artificial intelligence analysis on the iFIND platform, this method can be further optimized to not only detect mutations but also precisely locate mutation sites, providing stronger support for in-depth research on tuberculosis drug resistance mechanisms and precision medicine.

(Specific peak of rpoB S531L)

The molecular beacon melting curve method adopted by iFIND provides an efficient, accurate, and fully covering solution for rifampicin resistance detection in tuberculosis. Compared with traditional methods, its rapidity, high sensitivity, and specificity significantly improve the detection rate of drug-resistant mutations, making it especially suitable for widespread use in primary medical institutions.

Rocgene Warmly Invites You

The 2025 National Tuberculosis Academic Conference of the Chinese Medical Association will be grandly held in Beijing Capital International Convention Center on August 8–9, 2025. Rocgene will demonstrate the iFIND® Fully Automatic Molecular Detection Platform at Booth No. 030. We welcome you to visit our booth for communication and discussion!