Name: Video: Gastric Fluid Sampling, DNA Extraction, and qPCR for H. pylori Detection and its Antibiotic Resistance
Uploaded: 2023-07-28T13:20:00
Description: 450 Views. Gastric Fluid Sampling, DNA Extraction, and qPCR for H. pylori Detection and its Antibiotic Resistance

gastric fluid sampling, dna extraction, and qpcr for h. pylori detection and its antibiotic resistance

qPCR-Based Rapid, Noninvasive Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

H. pylori is a spiral-shaped, highly motile, gram-negative bacterium that mainly lives in the pylorus region of the stomach1. It&#160;is a common pathogen that infects nearly 50% of the global population2. Most people with H. pylori infection have no clinical manifestations, and most develop different diseases after several years of infection, including chronic gastritis, peptic ulcers, gastric ulcers, and gastric cancer3. In several studies based on different populations, the efficacy of eliminating H. pylori for preventing stomach cancer and precancerous lesions has been demonstrated4,5. Therefore, the World Health Organization (WHO) International Agency for Research on Cancer has advised H. pylori eradication as a preventative measure6.
The use of noninvasive methods to identify H. pylori infection is a key component of treatment for most individuals with asymptomatic dyspepsia. The urea breath test (UBT), H. pylori fecal antigen test (SAT), and serological testing are popular noninvasive techniques. Among these, the UBT is the least intrusive and most accurate procedure available. UBT uses urease, abundantly present in H. pylori, to hydrolyze isotopically labeled urea into ammonia and carbon dioxide (13C or 14C). In contrast, the immunochromatographic assay (ICA)7 is convenient, simple, and noninvasive for sampling. However, the accuracy of the test is affected by several factors, such as the quality of the stool sample, the temperature, and the interval between the sample collection and testing. Another test based on the immune response is the serum H. pylori antibody test, which detects antibodies in a patient&#39;s serum. However, this test is not suitable for post-treatment analysis since the antibodies remain long after the bacteria have been cleared8. Another major drawback is that these methods only diagnose H. pylori infection and do not allow for drug resistance testing to guide sensitivity-based treatment.
For invasive testing methods, gastric biopsy tissue needs to be taken by endoscopy and then subjected to histology, the urease rapid test, and bacterial culture. These testing methods are also very limited due to several factors. Currently, these techniques are limited to elderly patients, patients at high risk for precancerous or malignant disease, and patients who have failed first-line therapy for gastroesophageal reflux disease or H. pylori infection9. Secondly, due to the unique growth characteristics of H. pylori, the success rate of bacterial culture only reaches 50%10. Thus, molecular detection methods offer new hope to overcome the high demands of invasive detection methods and guide sensitivity-based treatment. Among molecular detection methods, quantitative PCR has evolved tremendously in recent years. qPCR, unlike traditional PCR, does not require gel electrophoresis and accurately quantifies DNA/RNA in samples by adding primers and probes at the annealing stage. qPCR kits for the detection of H. pylori infection and drug resistance are now commercially available. Nevertheless, each method has its limitations; therefore, a patient&#39;s clinical diagnosis and treatment should be considered in conjunction with their symptoms, signs, history, other laboratory tests, and response to treatment.
Currently, the primary method of treating H.pylori infections is taking antibiotics, but lately, it is becoming increasingly difficult to treat these infections due to the rise in antibiotic resistance. Subsequently, a significant decline in H. pylori treatment efficacy has been observed globally, making H. pylori eradication a major public health issue11.
Clarithromycin and levofloxacin are the two broad-spectrum antibiotics used to treat infections caused by H.pylori,&#160;but several studies have reported widespread resistance against these two drugs in H.pylori isolates. A2143G, A2142G, and A2142C are three of the numerous point mutations found in the 2.9 kb 23S rRNA gene that result in clarithromycin resistance by preventing the macrolide from binding. At the same time, the mutation loci of the levofloxacin resistance gene are mainly located in the six mutation sites (A260T, C261A, T261G, G271A, G271T, A272G) of the gyrA gene12. The discovery of these resistance mechanisms based on genetic mutations has led to a gradual shift in the detection of H. pylori through cultural-based studies to molecular testing.
Overall, there is an urgent clinical need for a noninvasive, effective, and simultaneous diagnostic method for the detection of H. pylori infections and drug resistance. We adopted a combined string test and qPCR method to overcome the difficulties of sampling and achieve the goal of the simultaneous detection of H. pylori infection and drug resistance using different primer probes.

Author Spotlight: Detection and Treatment of Helicobacter pylori Infection 

Quantitative Polymerase Chain Reaction (qPCR)-Based Rapid Diagnosis of Helicobacter pylori Infection and Antibiotic Resistance

Watch this Scientific Journal Video about Gastric Fluid Sampling, DNA Extraction, and qPCR for H. pylori Detection and its Antibiotic Resistance at JoVE.com

Gastric Fluid Sampling, DNA Extraction, and qPCR for H. pylori Detection and its Antibiotic Resistance  

Gastric Fluid Sampling, DNA Extraction, and qPCR for H. pylori Detection and its Antibiotic Resistance

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Research

JoVE Journal

Immunology and Infection

450 Views. Gastric Fluid Sampling, DNA Extraction, and qPCR for H. pylori Detection and its Antibiotic Resistance

Video: Gastric Fluid Sampling, DNA Extraction, and qPCR for H. pylori Detection and its Antibiotic Resistance  

Helicobacter pylori is a major human pathogen that&#160;infects approximately half of the global population and is becoming a serious health threat due to its increasing antibiotic resistance. It is the causative agent of chronic active gastritis, peptic ulcer disease, and gastric cancer and has been classified as a Group I Carcinogen by the International Agency for Research on Cancer. Therefore, the rapid and accurate diagnosis of H. pylori and the determination of its antibiotic resistance are important for the efficient eradication of this bacterial pathogen. Currently, H. pylori diagnosis methods mainly include the urea breath test (UBT), the antigen test, the serum antibody test, gastroscopy, the rapid urease test (RUT), and bacterial culture. Among them, the first three detection methods are noninvasive, meaning they are easy tests to conduct. However, bacteria cannot be retrieved through these techniques; thus, drug resistance testing cannot be performed. The last three are invasive examinations, but they are costly, require high skills, and have the potential to cause damage to patients. Therefore, a noninvasive, rapid, and simultaneous method for H. pylori detection and drug resistance testing is very important for efficiently eradicating H. pylori in clinical practice. This protocol aims to present a specific procedure involving the string test in combination with quantitative polymerase chain reaction (qPCR) for the rapid detection of H. pylori infection and antibiotic resistance. Unlike bacterial cultures, this method allows for easy, rapid, noninvasive diagnosis of H. pylori infection status and drug resistance. Specifically, we used qPCR to detect rea for H. pylori infection and mutations in the 23S rRNA and gyrA genes, which encode resistance against clarithromycin and levofloxacin, respectively. Compared to routinely used culturing techniques, this protocol provides a noninvasive, low-cost, and time-saving technique to detect H. pylori infection and determine its antibiotic resistance using qPCR.

The protocol presents a noninvasive method for the rapid diagnosis of Helicobacter pylori stomach infections through the string test and determines its antibiotic resistance to clarithromycin and levofloxacin using quantitative polymerase chain reaction (qPCR).

Helicobacter pylori is a major human pathogen that&#160;infects approximately half of the global population and is becoming a serious health threat due to ...