Establishment and Evaluation of an Overlap Extension Polymerase Chain Reaction Technique for the Rapid and Efficient Detection of Drug Resistance in Mycobacterium tuberculosis | Infectious diseases of poverty

Design and implementation of the study

This was a diagnostic test evaluation for the detection of Mr. tuberculosis drug resistance conducted at a tertiary hospital specializing in infectious diseases, i.e. Chongqing Public Health Medical Center in China. The test method is SOE PCR with Sanger sequencing, and reference methods include phenotypic DST and GeneXpert MTB/RIF. 108 clinics Mr. tuberculosis isolates from December 2018 to April 2019 were selected to compare the SOE PCR test with the phenotypic DST to detect resistance to RFP, INH and EMB. Of these samples, 56 isolates that had GeneXpert MTB/RIF results were used to compare the SOE PCR assay to detect RFP resistance.

Isolate selection criteria

Mr. tuberculosis isolates with different resistance properties were selected. The selection criteria were as follows: (a) The strains were isolated from the sputum of tuberculosis patients and positively identified as Mr. tuberculosis according to standard laboratory procedures [16]; (b) Phenotypic DST results for RFP, INH and EMB resistance were complete and traceable, among which samples with GeneXpert MTB/RIF results would be compared with SOE PCR test; (c) Strains should have different resistance properties, including isolates susceptible to drugs and those resistant to one or more drugs.

Reference methods

Phenotypic DST and GeneXpert MTB/RIF were used as reference methods as they were considered the standard methods for the diagnosis of drug-resistant TB [3]. Phenotypic DST was performed according to standard guidelines using the proportional L–J method, for RFP, INH and EMB [16]. GeneXpert MTB/RIF was performed according to the standard protocol [5].

Reagents and instrumentation for the SOE PCR test

10× buffer, dNTPs and hot start Taq polymerase used in the PCR reaction were purchased from Nanjing Nazyme Biotech Company (Nanjing, China), Lowenstein–Jensen medium was purchased from Zhuhai Yinke Company (Zhuhai, China), and primers were synthesized by Beijing Tsingke Biotech Co., Ltd., Chongqing Branch (Chongqing, China).

The main laboratory instruments used in this study included Eppendorf ThermoMixer C metal bath (Eppendorf AG, Germany), ETC811 PCR instrument (Eastwin Life Sciences Inc; Beijing, China), DYY-16D electrophoresis instrument ( Beijing Liuyi Instruments; Beijing, China), ChampGel Gel Imager (China Saizhi Company; Beijing, China) and GeneXpert MTB/RIF Assay (Cepheid; Sunnyvale, CA, USA).

Primers for the SOE PCR test

Using the DNA sequence of the reference strain from the laboratory of Mr. tuberculosis (H37Rv) as a template, we designed PCR primers whose enhancer products would contain the mutation sites of the rpoB, embB, katGand inha promoters. These genes confer resistance to RFP, INH and EMB. The specific sequences are shown in Table 1.

Table 1 Primer sequences for the SOE PCR assay

The principle of the SOE PCR test

Four main gene fragments related to drug resistance i.e. rpoB, embB, catGand the inha promoter, were ligated and amplified into an approximately 700 base pair (bp) fusion fragment in a single reaction tube, after which the four drug resistance-related gene fragments were sequenced via Sanger sequencing in a single read, greatly speeding up the detection of the presence of drug resistance mutations that may exist in a Mr. tuberculosis isolate.

There were three different complementary sequences overlapping between the primers rpoB_Rev (1R) and embB_For (2F), between primers embB_Inv (2R) and catG_For (3F), and between primers catG_Rev (3R) and inha_For (4F) respectively, as shown in different colors in Fig. 1. In the SOE PCR reaction system, the concentrations of primers 1F and 4R are higher than those of the other six primers, while the concentrations of primers 2R and 3F are higher than that of primers 1R, 2F, 3R and 4F.

Fig. 1

Block diagram of the SOE PCR. SOE PCR: Gene splicing by overlap extension PCR

At the initial stage of the SOE PCR reaction, double-stranded DNA fragments of rpoB, embB, katG, and inha were produced. As the reaction cycles increase, the 1R, 2F, 3R, 4F primers are depleted and exhausted, and the single-stranded DNA fragments of rpoB, embB, katG, and inha are produced. During this process, there is a probability of gene splicing between the fragments rpoB and embBand between the fragments catG and inha.

Then, as the reaction cycles increase, the 2R and 3F primers are also depleted and exhausted, and the single-stranded DNA fragments of rpoB-embB and spliced katG-inhA are produced. During this process, there is a probability of gene splicing between the spliced ​​rpoB-embB and the spliced ​​katG-inhA.

Once a small amount of rpoB-embB-katG-inhA fusion fragment is produced in the SOE PCR system, it can then be used as a template to be amplified by the 1F and 4R primers at both ends. The principle of SOE PCR in this study is shown in Fig. 1.

Detection of drug resistance in clinical isolates of M. tuberculosis using the SOE PCR test

H37Rv, the reference strain of the laboratory of Mr. tuberculosis, which is susceptible to all anti-tuberculosis drugs and does not have genetic mutations identifiable as drug resistance genes, was used as a wild-type control. Crude genomic DNA was isolated from freshly cultured bacteria via the rapid boil method. Briefly, bacterial cells were suspended with Tris-EDTA buffer (pH 8.0). Then 100 μl of suspensions at a concentration of 108 cells/ml were heated at 100°C for 30 min. Cell debris was removed by centrifugation at 12,000 g for 5 min. Genomic DNA in the supernatant was then used as PCR template.

DNA extracted from 108 clinics Mr. tuberculosis isolates was used for the amplification of rpoB-embB-katG-inhA fusion fragment via SOE PCR technique, and physiological saline solution was used as a negative control. Since natural rpoB-embB-katG-inhA fusion fragments do not exist in nature, it was not applicable to set up a positive control in this case. The 50 μl PCR reaction system contained 5.0 μl 10× buffer, 5.0 μl 10 μmol/l primers, 5.0 μl template DNA, 1.0 μl 2.5 mol/ l of dNTP, 0.2 μl of 5 U/μl Taq polymerase, and 33.8 μl of ddH2O. The PCR procedure was defined as follows: Step 1: 95°C for 5 min; Step 2: 95°C for 10s; Step 3: 68°C, reduce 0.5°C for each cycle for 10s; Step 4: 72°C for 30s; Step 5: 95°C for 10s, Step 6: 59°C for 10s, Step 7: 72°C for 30s; Stages 2 to 4: 30 cycles and Stages 5 to 7: 40 cycles. Thus, the total duration of the SOE PCR amplification process would be approximately 2 h.

the rpoB-embB-katG-inhA The fusion fragment obtained by SOE PCR amplification was sequenced using the TBseq primer. This stage of the process is likely to consume less than 5 hours. Sequencing results rpoB-embB-katG-inhA the fusion fragments of the 108 clinical isolates, along with the DNA sequence of the standard strain (H37Rv), were then imported into MegAlign for comparative analysis to search for associated drug resistance mutations. The DNA sequence of rpoB (1–273 bp)-embB (274–420 bp)-catG (421–564 bp)-inha (565-722 bp) fusion fragment obtained by the SOE PCR technique is shown in Supplementary File 1. The amino acid sequence associated with the rpoB-embB-katG-inhA The sequence of the standard strain H37Rv fusion DNA fragment is shown in Supplementary File 2 (inha promoter does not encode any amino acid).

The principle of mutational analysis is as follows:

  1. (1)

    Major mutation sites in the rpoB amino acid sequence included rpoB 511, 513, 515, 516, 518, 519, 526, 531, and 533, corresponding to amino acid mutation sites 51, 53, 55, 56, 58, 59, 66, 71 and 73 in the amino acid sequence rpoB-embB-katG-inhA. Table 2 illustrates the corresponding relationship of major mutation sites between the amino acid sequence of rpoB and the amino acid sequence of the fusion fragment.

  2. (2)

    Amino acid residue site 306 in embB corresponded to residue site 116 of the fusion fragment, the mutation of which can cause EMB resistance.

  3. (3)

    The site at amino acid residue 315 in katG corresponded to the site at amino acid residue 153 of the fusion fragment, the mutation of which can cause INH resistance.

  4. (4)

    Site 15 and site 8 of the inhA promoter DNA sequence corresponded to site 653 and site 660 of the inhA promoter DNA sequence. rpoB-embB-katG-inhA fusion fragment, and mutations at these two sites cause INH resistance.

Table 2 Corresponding relationship of major mutation sites between rpoB amino acid sequence and fusion fragment amino acid sequence

Data analysis

An appropriately trained investigator used a 4-grid chart to perform a Kappa identity test and to calculate sensitivity, specificity, positive predictive value, negative predictive value, concordance, and Kappa value, comparing the SOE PCR method results with phenotypic DST or GeneXpert VTT/RIF test results. Kappa values ​​below 0.2, 0.21-0.40, 0.41-0.60, 0.61-0.80, and above 0.8 are considered mild, medium, moderate agreement, respectively. , substantial and nearly perfect [17]. Data analysis was cross-checked by another qualified investigator and a statistician from the School of Biomedical Engineering, Capital Medical University, Beijing, China.

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